Methods for altering body composition

ABSTRACT

The present invention relates to compositions and methods for altering body composition in a subject, wherein the alteration of body composition is an increase in muscle mass and a reduction of fat mass simultaneously. The present invention also relates to compositions and methods for reducing fat mass in a subject. The compositions and methods also increase muscle volume and lean body mass in the subject. The present invention also relates to compositions that comprise a GDF8 inhibitor and an Activin A inhibitor and the use of such compositions to treat diseases and disorders characterized by increased fat mass, and/or decreased muscle volume.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/637,017, filed 1 Mar. 2018, the entire contents of which areincorporated herein by reference.

SEQUENCE LISTING

This application includes a Sequence Listing in electronic formatentitled “Sequence-Listing-40848-091USU1”, which was created on 26 Feb.2019 and which has a size of 288 kilobytes (KB) (295,202 bytes). Thecontents of txt fie “Sequence-Listing-40848-091USU1” are incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for alteringbody composition in a subject. The compositions and methods also reducefat mass in a subject. The compositions and methods also increase musclevolume and/or lean body mass in the subject. More specifically, theinvention relates to compositions that comprise a GDF8 inhibitor and anActivin A inhibitor and the use of such compositions to treat diseasesand disorders characterized by increased fat mass, and/or decreasedmuscle volume or decreased lean body mass.

BACKGROUND

Growth and differentiation factor-8 (GDF8, also known as myostatin), isa secreted ligand belonging to the transforming growth factor-β (TGF-β)superfamily of growth factors. GDF8 plays a central role in thedevelopment and maintenance of skeletal muscle, acting as a negativeregulator of myogenesis and skeletal muscle mass. Myostatin mutations(including knockout) translate into phenotypes that are predominantlyincreased muscle mass, but can be made up of variations in muscling(more muscle fibers), muscle fiber composition (greater cross-sectionalarea of muscle fibers), increased protein/DNA ratio, and other.

Antibodies to GDF8 and therapeutic methods are disclosed in, e.g., U.S.Pat. No. 8,840,894. Anti-GDF8 antibodies are also mentioned in, e.g.,U.S. Pat. Nos. 6,096,506; 7,320,789; 7,261,893; 7,807,159; 7,888,486;7,635,760; 7,632,499; in US Patent Appl. Publ. Nos. 2006/0263354;2007/0178095; 2008/0299126; 2010/0166764; 2009/0148436; andInternational Patent Appl. Publ. Nos. WO2004/037861; WO2007/047112; WO2010/070094.

Activins belong to the transforming growth factor-beta (TGF-β)superfamily and exert a broad range of biological effects on cellproliferation, differentiation, metabolism, homeostasis, and apoptosis,as well as immune response and tissue repair. Activin A is adisulfide-linked homodimer (two beta-A chains) that binds to andactivates heteromeric complexes of a type I (Act RI-A and Act RI-B) anda type II (Act RII-A and Act RII-B) serine-threonine kinase receptor.

Antibodies to Activin A and uses thereof are disclosed in, e.g., U.S.Pat. Nos. 8,309,082; 9,718,881; and International Patent Appl. Publ. No.WO2008/031061.

Compositions comprising an anti-GDF8 antibody and an anti-Activin Aantibody and therapeutic methods are disclosed in, e.g., U.S. Pat. No.8,871,209.

Obesity is a global problem for over a third of the world population. Inthe United States of America, the average obesity rate is over 20%. Thecosts of obesity-related illness are staggering, amounting to $190.2billion, roughly 21% of annual medical costs in the U.S. Obesity is anepidemic disease characterized by chronic low-grade inflammationassociated with dysfunctional (elevated) fat mass. In Framingham HeartStudy participants, abdominal adiposity was associated with incidentcardiovascular disease (CVD) after adjustments for clinical risk factorsand overall adiposity. Britton JACC 2013 62; 921. Abdominal visceral fataccumulation was positively associated with the progression of coronarynoncalcified plaque. Imai Atherosclerosis 2012. Because high fat mass isassociated with such serious conditions as congestive heart failure,high blood pressure/hypertension, pulmonary embolism, osteoarthritis,lymphedema, gastro-esophageal reflux disease, chronic renal failure,cancer, fatty-liver disease, and even depression, there remains a needfor therapies that reduce total fat and/or android fat mass in subjects.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention is directed to a method for altering bodycomposition in a subject, i.e., increase muscle mass and decreasing fatmass, comprising administering a first composition comprising aneffective amount of a GDF8 inhibitor and a second composition comprisingan effective amount of an Activin A inhibitor to the subject. In anotheraspect, the invention is directed to a method for inducing a reductionin fat mass in a subject comprising administering an effective amount ofa composition comprising a GDF8 inhibitor and an Activin A inhibitor tothe subject.

In another aspect, the invention is directed to a method for treating adisease or disorder characterized by or associated with increased fatmass, the method comprising administering to a subject in need thereof afirst composition comprising an effective amount of a GDF8 inhibitor anda second composition comprising an effective amount of an Activin Ainhibitor. In another aspect, the invention is directed to a method fortreating a disease or disorder characterized by or associated withincreased fat mass, the method comprising administering to a subject inneed thereof an effective amount of a composition comprising a GDF8inhibitor and an Activin A inhibitor. In one aspect, the invention isdirected to a method for altering body composition in a subject, i.e.,increase muscle mass and decreasing fat mass, comprising administeringan effective amount of a GDF8 inhibitor and an effective amount of anActivin A inhibitor to the subject, wherein there is an insignificantchange in total body mass. Thus, in one aspect of the invention, asubject administered an effective amount of a GDF8 inhibitor and aneffective amount of an Activin A inhibitor will experience an increasein muscle mass concurrently with a decrease in fat mass, leading to aminimal and/or insignificant change in total body mass.

In one aspect, the invention is directed to a use of a GDF8 inhibitorand/or an Activin A inhibitor in the preparation of a medicament forachieving a reduction in fat mass in a subject. In another aspect, theinvention is directed to a use of a GDF8 inhibitor and/or an Activin Ainhibitor in the preparation of a medicament for treating a disease ordisorder associated with increased fat mass in a subject.

In some embodiments, a GDF8 inhibitor is provided for use in a methodfor treating a disease or disorder characterized by increased fat mass,wherein the method comprises administrating to a subject the GDF8inhibitor and an Activin A inhibitor.

In some embodiments, an Activin A inhibitor is provided for use in amethod for treating a disease or disorder characterized by increased fatmass, wherein the method comprises administrating to a subject theActivin A inhibitor and a GDF8 inhibitor.

In some embodiments, a non-therapeutic method is provided for decreasingfat mass in a subject, the method comprising administering to thesubject an Activin A inhibitor and a GDF8 inhibitor.

In one embodiment of a method according to the invention, the effectiveamount of a GDF8 inhibitor comprises a dosing regimen selected from thegroup consisting of at least 0.1 mg/kg to about 10 gm/kg, 1 mg/kg toabout 1 gm/kg, and 10 mg/kg to 100 mg/kg. In a further embodiment of amethod according to the invention, the effective amount of a GDF8inhibitor comprises a dosing regimen selected from the group consistingof a single dose of about 0.01 to about 20 mg/kg body weight, about 0.1to about 10 mg/kg body weight, and about 0.1 to about 5 mg/kg bodyweight.

In another embodiment of a method according to the invention, theeffective amount of an Activin A inhibitor comprises a dosing regimenselected from the group consisting of at least 0.1 mg/kg to about 10gm/kg, 1 mg/kg to about 1 gm/kg, and 10 mg/kg to 100 mg/kg. In a furtherembodiment of a method according to the invention, the effective amountof an Activin A inhibitor comprises a dosing regimen selected from thegroup consisting of a single dose of about 0.01 to about 20 mg/kg bodyweight, about 0.1 to about 10 mg/kg body weight, and about 0.1 to about5 mg/kg body weight.

In one embodiment of a method according to the invention, the effectiveamount of a GDF8 inhibitor is 6 mg/kg body weight and the effectiveamount of an Activin A inhibitor is 3 mg/kg body weight. In oneembodiment of a method according to the invention, the effective amountof a GDF8 inhibitor is 6 mg/kg body weight and the effective amount ofan Activin A inhibitor is 10 mg/kg body weight.

In one embodiment of a method according to the invention, the firstcomposition is formulated for intravenous, subcutaneous, or oraladministration. In another embodiment of a method according to theinvention, the second composition is formulated for intravenous,subcutaneous, or oral administration. In certain embodiments of a methodaccording to the invention, the first and second compositions areadministered concurrently or sequentially to the subject.

In one embodiment of a method according to the invention, the first andsecond compositions are combined into a third composition prior toadministration. In a further embodiment, the third composition isformulated for intravenous, subcutaneous, or oral administration.

In one embodiment, a method according to the invention further comprisesmeasuring total fat mass in the subject before administration. Inanother embodiment, a method according to the invention furthercomprises measuring total fat mass in the subject after administration,and administering the first and second composition until the subject hasa reduction in total fat mass of at least 2% to 8%, 2.5% to 6%, 3% to4%, or at least 2.0%, at least 2.5%, at least 3.0%, or at least 3.5%, ormore.

In one embodiment, a method according to the invention further comprisesmeasuring android fat mass in the subject before administration. Inanother embodiment, a method according to the invention furthercomprises measuring android fat mass in the subject afteradministration, and administering the first and second composition untilthe subject has a reduction in android fat mass of at least 2% to 8%,2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%, at least 3.0%, orat least 3.5%, or more.

In one embodiment, a method according to the invention further comprisesmeasuring subcutaneous adipose tissue volume in the subject beforeadministration. In another embodiment, a method according to theinvention further comprises measuring subcutaneous adipose tissue volumein the subject after administration, and administering the first andsecond composition until the subject has a reduction in android fat massof at least 2% to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least2.5%, at least 3.0%, or at least 3.5%, or more.

In some embodiments, a method is provided comprising administering to asubject in need thereof an effective amount of a GDF8 inhibitor and aneffective amount of a Activin A inhibitor, wherein the GDF8 inhibitorand the Activin A inhibitor are co-administered within 48 hours or less,24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, or1 hour or less.

In some embodiments, a method is provided comprising administering to asubject in need thereof an effective amount of a GDF8 inhibitor and aneffective amount of a Activin A inhibitor, wherein the subject exhibitsa decrease in total fat mass, android fat mass, and/or subcutaneousadipose tissue volume.

In some embodiments, a method is provided comprising administering to asubject in need thereof an effective amount of a GDF8 inhibitor and aneffective amount of a Activin A inhibitor, wherein the subject exhibitsa decrease in total fat mass, android fat mass, and/or subcutaneousadipose tissue volume after 4 weeks or more, or 8 weeks or more,following administration.

In some embodiments, a method is provided comprising administering to asubject in need thereof an effective amount of a GDF8 inhibitor and aneffective amount of a Activin A inhibitor, wherein the subject exhibitsa decrease in total fat mass, android fat mass, and/or subcutaneousadipose tissue volume, wherein the subject does not exhibit reducedthigh intramuscular adipose tissue volume.

In some embodiments, a method is provided comprising administering to asubject in need thereof an effective amount of a GDF8 inhibitor and aneffective amount of a Activin A inhibitor, wherein the subject exhibitsa decrease in total fat mass, android fat mass, and/or subcutaneousadipose tissue volume, wherein the subject does not exhibit reducedthigh intramuscular adipose tissue volume after 4 weeks or more, or 8weeks or more, following administration.

In some embodiments, a kit is provided comprising a first containercontaining a effective amount of a GDF8 inhibitor and a second containercontaining an effective amount of a specific Activin A inhibitor.

In some embodiments, the GDF8 inhibitor is an isolated antibody or anantigen-binding fragment thereof that specifically binds to GDF8.

In one embodiment of a method according to the invention, the GDF8inhibitor is an antibody or an antigen-binding fragment thereof thatspecifically binds to GDF8. In another embodiment, the antibody orantigen-binding fragment that specifically binds GDF8 comprises theheavy chain complementarity determining regions (HCDRs) of a heavy chainvariable region (HCVR) comprising SEQ ID NO:360, and the light chaincomplementarity determining regions (LCDRs) of a light chain variableregion (LCVR) comprising SEQ ID NO:368. In still another embodiment, theantibody or antigen-binding fragment that specifically binds GDF8comprises three HCDRs comprising SEQ ID NO:362, SEQ ID NO:364, and SEQID NO:366, and three LCDRs comprising SEQ ID NO:370, SEQ ID NO:372, andSEQ ID NO:374.

In some embodiments, the Activin A inhibitor is an isolated antibody oran antigen-binding fragment thereof that specifically binds to ActivinA.

In one embodiment of a method according to the invention, the Activin Ainhibitor is an antibody or antigen-binding fragment thereof thatspecifically binds Activin A. In another embodiment, the antibody orantigen-binding fragment that specifically binds Activin A comprises theheavy chain complementarity determining regions (HCDRs) of a heavy chainvariable region (HCVR) comprising SEQ ID NO:553, and the light chaincomplementarity determining regions (LCDRs) of a light chain variableregion (LCVR) comprising SEQ ID NO:537. In still another embodiment, theantibody or antigen-binding fragment that specifically binds Activin Acomprises three HCDRs comprising SEQ ID NO:555, SEQ ID NO:557, and SEQID NO:559, and three LCDRs comprising SEQ ID NO:539, SEQ ID NO:541, andSEQ ID NO:543.

In one embodiment of a method according to the invention, the effectivedose of the Activin A inhibitor is selected from the group comprisingbetween 100% to 200% of the effective dose of the GDF8 inhibitor,between 100% and 250% of the effective dose of the GDF8 inhibitor,between 100% and 300% of the effective dose of the GDF8 inhibitor, andbetween 100% and 400% by weight of the effective dose of the GDF8inhibitor.

In another embodiment of a method according to the invention, the weightratio of the effective dose of the Activin A inhibitor to effective doseof the GDF8 inhibitor is from 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 3:1to 1:3, or about 2:1 to 1:2. In another embodiment of a method accordingto the invention, the weight ratio of the effective dose of the ActivinA inhibitor is about 1.5 to 2.0 times as large by weight as the amountthe GDF8 inhibitor.

In one embodiment of a method according to the invention, the GDF8inhibitor is a bispecific antibody or antigen-binding fragment thereofthat specifically binds GDF8 and also specifically binds Activin A. Inanother embodiment, the Activin A inhibitor is a bispecific antibody orantigen-binding fragment thereof that specifically binds Activin A andalso specifically binds GDF8.

In one embodiment of a method according to the invention, the reductionof fat mass in the subject is a reduction in total fat mass as measuredby DXA (Dual-energy X-ray absorptiometry). In another embodiment of amethod according to the invention, the reduction of fat mass in thesubject is a reduction in android fat mass as measured by DXA(Dual-energy X-ray absorptiometry).

In one embodiment of a method according to the invention, the reductionof fat mass in the subject is a reduction in subcutaneous adipose tissuevolume as measured by MRI (Magnetic Resonance Imaging).

In one embodiment of a method according to the invention, the subjectexperiences an increase in muscle volume. The muscle volume may be thighmuscle tissue volume, for example, as measured by MRI. In someembodiments, the muscle volume may be thigh muscle tissue volume, forexample, as measured by MRI. In some embodiments, the thigh musclevolume may be thigh muscle tissue volume including intramuscular adiposetissue and large vessels, or thigh muscle tissue volume excludingintramuscular adipose tissue and large vessels, for example, as measuredby MRI.

In one embodiment of a method according to the invention, the subjectexperiences an increase in total lean mass. The total lean mass may bemeasured by DXA (dual x-ray absorptiometry.

In one embodiment of a method according to the invention, the subjectexperiences an increase in appendicular lean body mass. The appendicularlean body mass may be measured by DXA, and, for example, calculated byaLBM equation.

In one embodiment of a method according to the invention, the subjectexperiences a decrease in total fat mass, for example, as measured byDXA.

In one embodiment of a method according to the invention, the subjectexperiences a decrease in android fat mass, for example, as measured byDXA.

In one embodiment of a method according to the invention, the subjectexperiences a decrease in subcutaneous adipose tissue volume, forexample, as measured by DXA.

In one embodiment of a method according to the invention, the subjectexperiences a decrease in sum of fat mass of arms and legs, for example,as measured by DXA.

In one embodiment of the invention, the subject does not exhibit adecrease in thigh intramuscular adipose tissue volume, for example, asmeasured by MRI.

In one embodiment of the invention, the subject does not exhibit adecrease in total bone mineral density (BMD) mass, for example, asmeasured by DXA.

In one embodiment of the invention, the subject does not exhibit adecrease in total bone mineral content (BMC) mass, for example, asmeasured by DXA.

In one embodiment of the invention, the subject exhibits an increase intotal bone mineral content (BMC) mass, for example, as measured by DXA.

In another embodiment of a method according to the invention, thesubject does not have a muscle wasting condition or disease.

In some embodiments, a kit is provided for use in altering bodycomposition, decreasing fat mass, increasing lean mass, or treating adisease or disorder characterized by or associated with increased fatmass, the kit comprising a first container comprising a compositioncomprising an effective amount of a GDF8 inhibitor and a secondcontainer comprising a second composition comprising an effective amountof an Activin A inhibitor.

In some embodiments, a GDF8 inhibitor is provided for use in manufactureof a first composition for use as a medicament in a kit for alteringbody composition, decreasing fat mass, increasing lean mass, or treatinga disease or disorder characterized by or associated with increased fatmass, the kit further comprising a second composition comprising anActivin A inhibitor.

In some embodiments, an Activin A inhibitor is provided for use inmanufacture of a first composition for use as a medicament in a kit foraltering body composition, decreasing fat mass, increasing lean mass, ortreating a disease or disorder characterized by or associated withincreased fat mass, the kit further comprising a second compositioncomprising a GDF8 inhibitor.

In some embodiments, a GDF8 inhibitor is provided for use in manufactureof a first composition for use in a kit for altering body composition,decreasing fat mass, or increasing lean mass in a subject, the kitfurther comprising a second composition comprising an Activin Ainhibitor.

In some embodiments, an Activin A inhibitor is provided for use inmanufacture of a first composition for use in a kit for altering bodycomposition, decreasing fat mass, or increasing lean mass in a subject,the kit further comprising a second composition comprising a GDF8inhibitor.

In some embodiments, a first composition comprising a GDF8 inhibitor isprovided for use in altering body composition, decreasing fat mass,increasing lean mass, or treating a disease or disorder characterized byor associated with increased fat mass or decreased lean mass in asubject, wherein the subject has received a second compositioncomprising an Activin A inhibitor.

In some embodiments, a first composition comprising an Activin Ainhibitor is provided for use in altering body composition, decreasingfat mass, increasing lean mass, or treating a disease or disordercharacterized by or associated with increased fat mass or decreased leanmass in a subject, wherein the subject has received a second compositioncomprising a GDF8 inhibitor.

In some embodiments, a first composition comprising a GDF8 inhibitor isprovided for use in method for altering body composition, decreasing fatmass, increasing lean mass, or treating a disease or disordercharacterized by or associated with increased fat mass or decreased leanmass, the method further comprising administering a second compositioncomprising an Activin A inhibitor.

In some embodiments, a first composition comprising an Activin Ainhibitor is provided for use in method for altering body composition,decreasing fat mass, increasing lean mass, or treating a disease ordisorder characterized by or associated with increased fat mass ordecreased lean mass, the method further comprising administering asecond composition comprising an GDF8 inhibitor.

In some embodiments, a composition is provided comprising an Activin Ainhibitor and a GDF8 inhibitor for use in altering body composition,decreasing fat mass, increasing lean mass, or treating or preventing adisease or disorder characterized by or associated with increased fatmass or decreased lean mass.

Other embodiments of the present invention will become apparent from areview of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a bar graph depicting results of clinical studies insarcopenia patients 70 years of age or older receiving anti-GDF8antibody REGN1033 alone after 12 weeks as the percent change frombaseline of total lean mass in LS mean and SE compared to placebo.Patients receiving REGN1033 exhibited significantly increased total leanbody mass at 12 weeks at each of three dosing regimens when compared toplacebo (n=65). Patients receiving 100 mg anti-GDF8 antibody REGN1033Q4W S.C. exhibited a difference vs. placebo of 1.66% total lean mass(n=62, P=0.0077). Patients receiving 300 mg anti-GDF8 Q4W SC exhibited adifference vs. placebo of 1.78% total lean mass (n=64, P=0.0043).Patients receiving 300 mg Q2W SC exhibited a difference vs. placebo of2.29% total lean mass (n=59, P=0.0004).

FIG. 2A shows Table 1 with the ascending dose panels used in the studyof 48 healthy post-menopausal women according to Example 2. A singleintravenous dose of anti-GDF8 antibody REGN1033 and/or anti-Activin Aantibody REGN2477 was utilized. In the primary analyses, the placebo andhigh dose combination groups were pooled across panels, yielding 12subjects on placebo and 12 on the high dose combination, as shown byboxed regions in the table.

FIG. 2B shows a bar graph depicting thigh muscle volume (measured viaMRI) % change at week 8 after a single I.V. dose of anti-Activin Aantibody REGN2477 and/or anti-GDF8 antibody REGN1033 in healthypost-menopausal women. A significant increase in thigh muscle volume %change from placebo was exhibited by anti-GDF8 (6 mg/kg), anti-GDF8 (6mg/kg)+mid dose anti-Activin A (3 mg/kg), and anti-GDF8 (6 mg/kg)+highdose anti-Activin A (10 mg/kg) groups. (*nominal p<0.5 vs. placebo,****nominal p<0.0001 vs. placebo).

FIG. 2C shows a bar graph depicting total fat mass (measured via DXA) %change at week 8 after a single I.V. dose of anti-Activin A antibodyREGN2477 and/or anti-GDF8 antibody REGN1033 in healthy post-menopausalwomen. The numbers show changes from placebo. A significant decrease intotal fat mass % change was exhibited by the anti-GDF8 (6 mg/kg)+highdose anti-Activin A (10 mg/kg) group. (*nominal p<0.05 vs. placebo).

FIG. 3 shows a line graph depicting LS mean (SE) percent change frombaseline in thigh muscle volume by MRI at weeks 0, 4, and 8 after asingle I.V. dose of anti-Activin A antibody REGN2477 and/or anti-GDF8antibody REGN1033 in healthy post-menopausal women. At 4 weeks aftersingle I.V. dose, a significant increase in thigh muscle volume(measured by MRI excluding intramuscular adipose tissue) % change fromplacebo was exhibited by each of anti-GDF8 (6 mg/kg), anti-GDF8 (6mg/kg)+low dose anti-Activin A (1 mg/kg), anti-GDF8 (6 mg/kg)+mid doseanti-Activin A (3 mg/kg), and anti-GDF8 (6 mg/kg)+high dose anti-ActivinA (10 mg/kg) groups. At 8 weeks after single I.V. dose, a significantincrease in thigh muscle volume % change from placebo was exhibited inanti-GDF8 (6 mg/kg)+mid dose anti-Activin A (3 mg/kg), and anti-GDF8 (6mg/kg)+high dose anti-Activin A (10 mg/kg) groups. (*nominal p<0.05,**nominal p<0.001). N values for each group are shown in FIG. 2A.

FIG. 4 show line graphs depicting individual data for percent change(from baseline) in thigh muscle volume by MRI at weeks 0, 4, and 8 aftera single I.V. dose of anti-Activin A antibody REGN2477 and/or anti-GDF8antibody REGN1033 in healthy post-menopausal women as % change frombaseline in placebo, anti-GDF8 (6 mg/kg), high dose anti-Activin A (10mg/kg), anti-GDF8(6 mg/kg)+low dose anti-Activin A(1 mg/kg), anti-GDF8(6mg/kg)+mid dose anti-Activin A(3 mg/kg), and anti-GDF8(6 mg/kg)+highdose anti-Activin A (10 mg/kg) groups for multiple individuals.Increases in thigh muscle volume were consistently observed inindividual subjects following treatment with REGN2477+REGN1033 incombination. Within each treatment group, different lines indicatedifferent individuals.

FIG. 5 shows a line graph depicting appendicular lean (body) mass (i.e.,sum of lean tissue in the arms and legs) at weeks 0, 4, and 8 after asingle I.V. dose of anti-Activin A antibody REGN2477 and/or anti-GDF8antibody REGN1033 in healthy post-menopausal women as % percent changefrom baseline LS (least-squares) mean (SE) in six groups. N values foreach group are shown in FIG. 2A. After 4 weeks and 8 weeks, each of thethree combination dose groups, including anti-GDF8(6 mg/kg)+low doseanti-Activin A(1 mg/kg), anti-GDF8(6 mg/kg)+mid dose anti-Activin A(3mg/kg), and anti-GDF8(6 mg/kg)+high dose anti-Activin A (10 mg/kg)groups, exhibited significantly increased % change LS mean difference inappendicular lean mass compared to placebo (*nominal p<0.05, **nominalp<0.001). N values for each group are shown in FIG. 2A.

FIG. 6 shows a line graph depicting mean (SE=standard errors) total fatmass percent (as measured by DXA) change at weeks 0, 4, and 8 after asingle I.V. dose of anti-Activin A antibody REGN2477 and/or anti-GDF8antibody REGN1033 in healthy post-menopausal women in placebo, anti-GDF8(6 mg/kg), high dose anti-Activin A (10 mg/kg), anti-GDF8(6 mg/kg)+lowdose anti-Activin A(1 mg/kg), anti-GDF8(6 mg/kg)+mid dose anti-ActivinA(3 mg/kg), and anti-GDF8(6 mg/kg)+high dose anti-Activin A (10 mg/kg)groups. N values for each group are shown in FIG. 2A. The high dosegroup, (anti-GDF8(6 mg/kg)+high dose anti-Activin A (10 mg/kg),exhibited a significant reduction in total fat mass as percent change LSMean difference compared to placebo at week 4 and week 8 (*nominalp<0.05). Blockade of both Activin A and GDF8 led to reductions in totalfat mass, as assessed by DXA.

FIG. 7 shows a line graph depicting mean (SE) percent change in androidfat mass at weeks 0, 4, and 8 after a single I.V. dose of anti-Activin Aantibody REGN2477 and/or anti-GDF8 antibody REGN1033 in healthypost-menopausal women in six groups including placebo, anti-GDF8(6mg/kg), high dose (10 mg/kg) anti-Activin A, anti-GDF8(6 mg/kg)+low dose(1 mg/kg) anti-Activin A, anti-GDF8(6 mg/kg)+mid dose (3 mg/kg)anti-Activin A, and anti-GDF8 (6 mg/kg)+high dose (10 mg/kg)anti-Activin A groups. The high dose REGN1033+REGN2477 group exhibitedsignificantly reduced percent change LS Mean difference in android fatmass by DXA compared to placebo at week 4 and week 8 (*nominal p<0.05).N values for each group are shown in FIG. 2A. Blockade of both Activin Aand GDF8 was also associated with decreases in android fat mass, asassessed by DXA.

FIG. 8 shows a line graph depicting LS mean percent change with SE inthigh muscle volume (excluding intramuscular adipose tissue and largevessels) at weeks 0, 4, and 8 after a single I.V. dose of anti-Activin Aantibody REGN2477 and/or anti-GDF8 antibody REGN1033 in healthypost-menopausal women in six groups including placebo, anti-GDF8(6mg/kg), high dose (10 mg/kg) anti-Activin A, anti-GDF8 (6 mg/kg)+lowdose (1 mg/kg) anti-Activin A, anti-GDF8(6 mg/kg)+mid dose (3 mg/kg)anti-Activin A, and anti-GDF8(6 mg/kg)+high dose (10 mg/kg) anti-ActivinA groups. N values for each group are shown in FIG. 2A. Compared withplacebo, REGN2477+REGN1033 medium and high groups exhibitedsignificantly increased mean % change in thigh muscle volume at 4 weeksand 8 weeks. (*nominal p<0.05, **nominal p<0.001).

FIG. 9 shows a line graph depicting LS mean percent change with SE intotal lean mass at weeks 0, 4, and 8 after a single I.V. dose ofanti-Activin A antibody REGN2477 and/or anti-GDF8 antibody REGN1033 inhealthy post-menopausal women for six groups including placebo,anti-GDF8(6 mg/kg), high dose (10 mg/kg) anti-Activin A, anti-GDF8 (6mg/kg)+low dose (1 mg/kg) anti-Activin A, anti-GDF8 (6 mg/kg)+mid dose(3 mg/kg) anti-Activin A, and anti-GDF8(6 mg/kg)+high dose (10 mg/kg)anti-Activin A groups. N values for each group are shown in FIG. 2A.Compared with placebo, REGN2477+REGN1033 medium and high dose groupsexhibited significantly increased % change in total lean mass comparedto placebo at 4 weeks and 8 weeks. (*nominal p<0.05).

FIG. 10 shows a line graph depicting LS mean percent change with SE inappendicular lean body mass (calculated via aLBM equation), in kg, atweeks 0, 4, and 8 after a single I.V. dose of anti-Activin A antibodyREGN2477 and/or anti-GDF8 antibody REGN1033 in healthy post-menopausalwomen in six groups including placebo, anti-GDF8, high dose (10 mg/kg)anti-Activin A, anti-GDF8+low dose (1 mg/kg) anti-Activin A,anti-GDF8+mid dose (3 mg/kg) anti-Activin A, and anti-GDF8+high dose (10mg/kg) anti-Activin A groups. N values for each group are shown in FIG.2A. REGN2477+REGN1033 treatment resulted in significantly increased %change in appendicular lean body mass calculated by aLBM equation)(kg)at each low, medium and high dose groups at 4 weeks and 8 weeks comparedwith placebo (*p<0.05, **p<0.001).

FIG. 11 shows a line graph depicting LS mean percent change with SE intotal fat mass, in kg, at weeks 0, 4, and 8 after a single I.V. dose ofanti-Activin A antibody REGN2477 and/or anti-GDF8 antibody REGN1033 inhealthy post-menopausal women in six groups including placebo, anti-GDF8(6 mg/kg), high dose (10 mg/kg) anti-Activin A, anti-GDF8+low dose (1mg/kg) anti-Activin A, anti-GDF8(6 mg/kg)+mid dose (3 mg/kg)anti-Activin A, and anti-GDF8 (6 mg/kg)+high dose (10 mg/kg)anti-Activin A groups. N values for each group are shown in FIG. 2A. Thehigh dose combination REGN2477+REGN1033 treatment group exhibitedsignificantly decreased % total fat mass: −3.92% (high dose group)compared with placebo −0.65% at 8 weeks. (*nominal p<0.05).

FIG. 12 shows a line graph depicting LS mean percent change with SE inthigh muscle volume, in cm³, (including intramuscular adipose tissue andlarge vessels) at weeks 0, 4, and 8 after a single I.V. dose ofanti-Activin A antibody REGN2477 and/or anti-GDF8 antibody REGN1033 inhealthy post-menopausal women in six groups including placebo,anti-GDF8, high dose (10 mg/kg) anti-Activin A, anti-GDF8+low dose (1mg/kg) anti-Activin A, anti-GDF8+mid dose (3 mg/kg) anti-Activin A, andanti-GDF8+high dose (10 mg/kg) anti-Activin A groups. N values for eachgroup are shown in FIG. 2A. The high and medium dose REGN2477+REGN1033treatment groups exhibited significantly increased % change in thighmuscle volume, including intramuscular adipose tissue and large vesselsin medium and high treatment groups at 4 weeks and 8 weeks compared toplacebo (*nominal p<0.05, **nominal p<0.001).

FIG. 13 shows a line graph depicting LS mean percent change with SE inappendicular lean mass (sum of lean mass of arms and legs), in kg, atweeks 0, 4, and 8 after a single I.V. dose of anti-Activin A antibodyREGN2477 and/or anti-GDF8 antibody REGN1033 in healthy post-menopausalwomen in six groups including placebo, anti-GDF8, high dose (10 mg/kg)anti-Activin A, anti-GDF8+low dose (1 mg/kg) anti-Activin A,anti-GDF8+mid dose (3 mg/kg) anti-Activin A, and anti-GDF8+high dose (10mg/kg) anti-Activin A groups. N values for each group are shown in FIG.2A. Appendicular lean mass was significantly increased as mean percentchange from placebo in each REGN2477+REGN1033 low, medium and hightreatment groups at 4 and 8 weeks (*nominal p<0.05, *nominal* p<0.001).

FIG. 14 shows a line graph depicting LS mean percent change with SE inandroid fat mass, in kg, at weeks 0, 4, and 8 after a single I.V. doseof anti-Activin A antibody REGN2477 and/or anti-GDF8 antibody REGN1033in healthy post-menopausal women in six groups including placebo,anti-GDF8, high dose (10 mg/kg) anti-Activin A, anti-GDF8+low dose (1mg/kg) anti-Activin A, anti-GDF8+mid dose (3 mg/kg) anti-Activin A, andanti-GDF8+high dose (10 mg/kg) anti-Activin A groups. N values for eachgroup are shown in FIG. 2A. The high dose REGN1033+REGN2477 groupexhibited significantly reduced % change in android fat mass by DXAcompared to placebo at week 4 and week 8 (*nominal p<0.05).

FIG. 15 shows a line graph depicting LS mean percent change with SE inthigh intramuscular adipose tissue volume, in cm³, at weeks 0, 4, and 8after a single I.V. dose of anti-Activin A antibody REGN2477 and/oranti-GDF8 antibody REGN1033 in healthy post-menopausal women in sixgroups including placebo, anti-GDF8, high dose (10 mg/kg) anti-ActivinA, anti-GDF8+low dose (1 mg/kg) anti-Activin A, anti-GDF8+mid dose (3mg/kg) anti-Activin A, and anti-GDF8+high dose (10 mg/kg) anti-Activin Agroups. N values for each group are shown in FIG. 2A. The high doseREGN1033+REGN2477 group exhibited increased thigh intramuscular adiposetissue volume as mean % change compared to placebo at 8 weeks. (*nominalp<0.05).

FIG. 16 shows a line graph depicting LS mean percent change with SE insum of intramuscular and perimuscular adipose tissue (IMAT) at weeks 0,4, and 8 after a single I.V. dose of anti-Activin A antibody REGN2477and/or anti-GDF8 antibody REGN1033 in healthy post-menopausal women insix groups including placebo, anti-GDF8, high dose (10 mg/kg)anti-Activin A, anti-GDF8+low dose (1 mg/kg) anti-Activin A,anti-GDF8+mid dose (3 mg/kg) anti-Activin A, and anti-GDF8+high dose (10mg/kg) anti-Activin A groups. N values for each group are shown in FIG.2A. The combination REGN1033+REGN2477 treatment groups were notsignificantly different than placebo in LS mean percent change with SEin sum of intramuscular and perimuscular adipose tissue (IMAT) at weeks4 and 8.

FIG. 17 shows a line graph depicting LS mean percent change with SE insubcutaneous adipose tissue volume, in cm³, at weeks 0, 4, and 8 after asingle I.V. dose of anti-Activin A antibody REGN2477 and/or anti-GDF8antibody REGN1033 in healthy post-menopausal women in six groupsincluding placebo, anti-GDF8, high dose (10 mg/kg) anti-Activin A,anti-GDF8+low dose (1 mg/kg) anti-Activin A, anti-GDF8+mid dose (3mg/kg) anti-Activin A, and anti-GDF8+high dose (10 mg/kg) anti-Activin Agroups. N values for each group are shown in FIG. 2A. TheREGN1033+REGN2477 low and medium treatment groups exhibitedsignificantly decreased % change in subcutaneous adipose tissue volumecompared to placebo at 8 weeks (*nominal p<0.05).

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular compositions, methods, andexperimental conditions described, as such compositions, methods, andconditions may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention will be limited only by the appended claims. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

As used herein, the term “about,” when used in reference to a particularrecited numerical value, means that the value may vary from the recitedvalue by no more than 1%. For example, as used herein, the expression“about 100” includes 99 and 101 and all values in between (e.g., 99.1,99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference in their entirety.

Antibodies and Antigen-Binding Fragments of Antibodies

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theantibodies (or antigen-binding portion thereof) may be identical to thehuman germline sequences, or may be naturally or artificially modified.An amino acid consensus sequence may be defined based on a side-by-sideanalysis of two or more CDRs.

Antibodies may be referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1M,” “H2aM,” “H4H”), followed by anumerical identifier (e.g. “10446”), followed by a “P,” “P2” or “N”suffix. Thus, according to this nomenclature, an antibody may bereferred to herein as, e.g., “H4H10446P2”. The H1M, H2M and H4H prefixeson the antibody designations used herein indicate the particular Fcregion isotype of the antibody. For example, an “H2aM” antibody has amouse IgG2a Fc, whereas an “H4H” antibody has a human IgG4 Fc. As willbe appreciated by a person of ordinary skill in the art, an antibodyhaving a particular Fc isotype can be converted to an antibody with adifferent Fc isotype (e.g., an antibody with a mouse IgG2a Fc can beconverted to an antibody with a human IgG4, etc.), but in any event, thevariable domains (including the CDRs) will remain the same, and thebinding properties are expected to be identical or substantially similarregardless of the nature of the Fc domain.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR thatis adjacent to or in frame with one or more framework sequences. Inantigen-binding fragments having a V_(H) domain associated with a V_(L)domain, the V_(H) and V_(L) domains may be situated relative to oneanother in any suitable arrangement. For example, the variable regionmay be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) or V_(L)-V_(L)dimers. Alternatively, the antigen-binding fragment of an antibody maycontain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody used in the present invention include: (i) V_(H)-C_(H)1;(ii) V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody used in the present inventionmay comprise a homo-dimer or hetero-dimer (or other multimer) of any ofthe variable and constant domain configurations listed above innon-covalent association with one another and/or with one or moremonomeric V_(H) or V_(L) domain (e.g., by disulfide bond(s)).

Full antibody molecules and antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantibody or antigen-binding fragment of an antibody will typicallycomprise at least two different variable domains, wherein each variabledomain is capable of specifically binding to a separate antigen or to adifferent epitope on the same antigen. Any multispecific antibody formatmay be adapted for use in the context of an antibody or antigen-bindingfragment described herein using routine techniques available in the art.

The antibodies used in the compositions and methods of the invention mayfunction through complement-dependent cytotoxicity (CDC) orantibody-dependent cell-mediated cytotoxicity (ADCC).“Complement-dependent cytotoxicity” (CDC) refers to lysis ofantigen-expressing cells by an antibody of the invention in the presenceof complement. “Antibody-dependent cell-mediated cytotoxicity” (ADCC)refers to a cell-mediated reaction in which nonspecific cytotoxic cellsthat express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland thereby lead to lysis of the target cell. CDC and ADCC can bemeasured using assays that are well known and available in the art.(See, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al.,Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998)).

The antibodies described herein may comprise or consist of humanantibodies and/or recombinant human antibodies, or fragments thereof.The term “human antibody”, as used herein, includes antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. Human antibodies may nonetheless include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibody molecules described herein may comprise or consist ofrecombinant human antibodies or antigen-binding fragments thereof. Theterm “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library,antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo. Recombinant antibodies may be used in an additional embodiment ofthe compositions and methods of the invention.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, tissueor cell in which the antibody naturally exists or is naturally producedis an “isolated antibody” for purposes of the invention. An isolatedantibody also includes an antibody in situ within a recombinant cell, aswell as an antibody that has been subjected to at least one purificationor isolation step. According to certain embodiments, an isolatedantibody may be substantially free of other cellular material and/orchemicals. Isolated antibodies may be used in an additional embodimentof the compositions and methods of the invention.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60, 70%, 80% or 90% w/w ofa protein sample, usually about 95%, and preferably over 99% pure.Protein purity or homogeneity may be indicated by a number of means wellknown in the art, such as polyacrylamide gel electrophoresis of aprotein sample, followed by visualizing a single polypeptide band uponstaining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “polypeptide analog or variant” as used herein refers to apolypeptide that is comprised of a segment of at least 25 amino acidsthat has substantial identity to a portion of an amino acid sequence andthat has at least one of the following properties: (1) specific bindingto GDF8 under suitable binding conditions, or (2) ability to block thebiological activity of GDF8. Typically, polypeptide analogs or variantscomprise a conservative amino acid substitution (or insertion ordeletion) with respect to the naturally occurring sequence. Analogstypically are at least 20 amino acids long, at least 50, 60, 70, 80, 90,100, 150 or 200 amino acids long or longer, and can often be as long asa full-length naturally-occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmutations of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton 1984 W. H. Freeman andCompany, New York; Introduction to Protein Structure (Branden & Tooze,eds., 1991, Garland Publishing, NY); and Thornton et at. 1991 Nature354:105, which are each incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (see, for example, Fauchere (1986) J. Adv. Drug Res.15:29; and Evans et al. (1987) J. Med. Chem. 30:1229, which areincorporated herein by reference. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo et al.(1992) Ann. Rev. Biochem. 61:387, incorporated herein by reference), forexample, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide.

As applied to polypeptides, the term “substantial identity” or“substantially identical” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least about 80% sequence identity, at least about90%, at least about 95%, at least about 98% or at least about 99%sequence identity. Preferably, residue positions that are not identicaldiffer by conservative amino acid substitutions. A “conservative aminoacid substitution” is one in which an amino acid residue is substitutedby another amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of similarity may be adjustedupwards to correct for the conservative nature of the substitution.Means for making this adjustment are well-known to those of skill in theart. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, hereinincorporated by reference. Examples of groups of amino acids that haveside chains with similar chemical properties include 1) aliphatic sidechains: glycine, alanine, valine, leucine and isoleucine; 2)aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; and 6) sulfur-containing side chainsare cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256:1443-45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000), supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially blastp or tblastn, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389 402, each of which is hereinincorporated by reference.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, at least about 20 residues, atleast about 24 residues, at least about 28 residues, or at least about35 residues. When searching a database containing sequences from a largenumber of different organisms, it is preferable to compare amino acidsequences.

In some embodiments, the invention relates to a method for altering thebody composition of a subject comprising administering a firstcomposition comprising an effective amount of a GDF8 inhibitor and asecond composition comprising an effective amount of an Activin Ainhibitor to the subject.

In some embodiments, the invention relates to a method for inducing areduction in fat mass in a subject comprising administering a firstcomposition comprising an effective amount of a GDF8 inhibitor and asecond composition comprising an effective amount of an Activin Ainhibitor to the subject.

In some embodiments, the invention relates to a method for inducing anincrease in muscle mass in a subject, the method comprisingadministering to a subject in need thereof a first compositioncomprising an effective amount of a GDF8 inhibitor and a secondcomposition comprising an effective amount of an Activin A inhibitor.

In some embodiments, the invention relates to a method for altering thebody composition of a subject comprising administering a compositioncomprising an effective amount of a GDF8 inhibitor and an effectiveamount of an Activin A inhibitor to the subject.

In some embodiments, the invention relates to a method for inducing areduction in fat mass in a subject comprising administering acomposition comprising an effective amount of a GDF8 inhibitor and aneffective amount of an Activin A inhibitor to the subject.

In some embodiments, the invention relates to a method for inducing anincrease in muscle mass in a subject, the method comprisingadministering to a subject in need thereof a composition comprising aneffective amount of a GDF8 inhibitor and an effective amount of anActivin A inhibitor.

In some embodiments, the subject is a human subject. The human subjectmay be an adult human subject. The subject may be a male or femalesubject. The subject may be a healthy subject. The subject may besuffering from unwanted fat mass. The subject may be suffering from, orat risk of suffering from, a disease and disorder characterized byincreased fat mass, and/or decreased muscle volume or decreased leanbody mass. The subject may be a post-menopausal female subject. Thesubject may be a male subject or female subject 40 years of age orolder, 50 years of age or older, 60 years of age or older, or 70 yearsof age or older.

In some embodiments, the invention relates to a method comprisingadministering a composition comprising a GDF8 inhibitor and acomposition comprising an Activin A inhibitor to a subject in needthereof for treating or preventing diseases and disorders characterizedby increased fat mass, and/or decreased muscle volume or decreased leanbody mass.

In some embodiments according to the invention, the subject has at leastone disease or disorder that may be associated with increased fat mass.In some embodiments, the disease or disorder may be selected from thegroup consisting of obesity, metabolic syndromes, nutritional disorders,high cholesterol, dyslipidemia, cardiovascular disease, cellulitis,cancer (including of the colon, esophagus, kidney, pancreas,gallbladder, breast, or endometrium), polycystic ovarian syndrome, gout,gallbladder disease, sleep apnea, respiratory disorder, asthma,osteoarthritis, cataract, congestive heart failure, enlarged heart, highblood pressure/hypertension, pulmonary embolism, lymphedema,gastro-esophageal reflux disease, hernia, chronic renal failure, urinaryincontinence, connective tissue diseases, and fatty-liver disease. Inanother embodiment, the disease or disorder may be sarcopenia.

GDF8 Inhibitors

The present invention includes methods for altering body composition,inducing a reduction in fat mass, and increasing lean mass in a subject,and methods for treating a disease or disorder characterized byincreased fat mass in a subject, comprising administering a compositioncomprising an effective amount of a GDF8 inhibitor to the subject.

The term “GDF8” (also referred to as “growth and differentiationfactor-8” and “myostatin”) means the protein having the amino acidsequence of SEQ ID NO:340 (mature protein). According to the presentinvention, GDF8-specific binding proteins specifically bind GDF8 but donot bind other ActRIIB ligands such as GDF3, BMP2, BMP4, BMP7, BMP9,BMP10, GDF11, Activin A, Activin B, Activin AB, Nodal, etc.

As used herein, a “GDF8 inhibitor” is any agent that binds to orinteracts with human GDF8 and interferes with or inhibits the normalbiological function of GDF8 in vitro or in vivo. Non-limiting examplesof categories of GDF8 inhibitors include small molecule GDF8antagonists, nucleic acid-based inhibitors of GDF8 expression oractivity (e.g., siRNA or antisense), peptide-based molecules thatspecifically interact with GDF8 (e.g., peptibodies), receptor moleculesthat specifically interact with GDF8, GDF8-binding scaffold molecules,proteins comprising a ligand-binding portion of a receptor thatspecifically binds GDF8, and anti-GDF8 aptamers or portions thereof. Ina preferred embodiment, a GDF8 inhibitor that can be used in the contextof the present invention is an anti-GDF8 antibody or antigen-bindingfragment thereof that specifically binds human GDF8. Anti-GDF8antibodies include neutralizing and/or blocking antibodies. Theinhibition caused by anti-GDF8 neutralizing and/or blocking antibodiesneed not be complete, as long as it is detectable using appropriateassays.

As used herein, the expression “anti-GDF8 antibody” also includesmultispecific antigen-binding molecules (e.g., bispecific antibodies),wherein at least one binding domain (e.g., “binding arm”) of themultispecific antigen-binding molecule specifically binds GDF8.

Exemplary anti-GDF8 antibodies that can be used in the compositions andmethods of the invention include, e.g., the fully-human anti-GDF8antibody H4H1657N2, also known as REGN1033, (e.g., an anti-GDF8 antibodycomprising the heavy and light chain variable regions having amino acidsequences SEQ ID NO: 360 and SEQ ID NO: 368, respectively, as set forthin U.S. Pat. No. 8,840,894). Other GDF8 antagonists that can be used inthe compositions and methods of the invention include anti-GDF8antibodies (e.g., the antibody designated 2_112_1 , e.g., having ATCCdeposit designation PTA-6574, or e.g., 2_ 112_K, e.g., having HCVR/LCVRamino acid sequences SEQ ID NOs: 620 and 621) as set forth in US2006/0263354 and U.S. Pat. No. 7,807,159; anti-GDF8 antibodies (e.g.,12A5-5, e.g., having HCVR/LCVR amino acid sequences of SEQ ID NO: 622and 623) as set forth in U.S. Pat. No. 8,999,343 and US Publication No.2013/0209489; anti-GDF8 antibodies (e.g., 10B3H8L5, e.g., havingHCVR/LCVR amino acid sequences of SEQ ID NO:624 and 625, and10B3H8L5-Fc-disabled) as set forth in US Publication No. 2013/0142788;anti-GDF8 antibodies (e.g., stamulumab/MYO-29, e.g., having HCVR/LCVRamino acid sequences of SEQ ID NOs: 626 and 627) as set forth in U.S.Pat. Nos. 8,940,874 and 7,261,893; anti-GDF8 antibodies (e.g.,RK22/PF-0625616, e.g., having HCVR/LCVR amino acid sequences of SEQ IDNO: 628 and 629) as set forth in U.S. Pat. No. 8,415,459; anti-GDF8antibodies (e.g., JA-16, e.g., having CDRs of HCVR amino acid sequenceof SEQ ID NO: 630) as set forth in U.S. Pat. No. 7,731,961; anti-GDF8antibodies (e.g., RK35, e.g., having HCVR/LCVR amino acid sequences ofSEQ ID NO: 631 and 632) as set forth in U.S. Pat. Nos. 8,496,934 or7,888,486, anti-GDF8 antibodies (e.g., OGD1.0.0, e.g., having HCVR/LCVRamino acid sequences of SEQ ID NO: 633 and 634) as set forth in U.S.Pat. No. 8,992,913; anti-GDF8 Fab molecules as set forth in EuropeanPatent No. 1 773 041 B1, and anti-GDF8 antibodies (e.g., C12, e.g.,having HCVR/LCVR amino acid sequences of SEQ ID NOs: 635 and 636,C12-N93H, and/or 510C2 having HCVR/LCVR amino acid sequences of SEQ IDNOs: 637 and 638) as set forth in, e.g., U.S. Pat. Nos. 7,635,760 and8,063,188, anti-GDF8 antibodies (e.g. 41C1E4/landogrozumab/LY2495655,e.g., having HCVR/LCVR amino acid sequences of SEQ ID NO: 639 and 640)as set forth in U.S. Pat. No. 7,632,499. In some embodiments, theanti-GDF8 antibody may have the full length heavy chain and full lengthlight chain amino acid sequences of landogrozumab, e.g., SEQ ID Nos 641and 642, respectively. In some embodiments, the anti-GDF8 antibody maycomprise three heavy chain CDRs (HCDRs) and three light chain CDRs(LCDRs) of landogrozumab, for example, by Chothia definition, accordingto SEQ ID Nos: 643/644/645/646/647/648, respectively.

In one embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises a heavy chain variable region (HCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NO:2, 18, 34,50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274,290, 306, 360, and 376, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises a light chain variable region (LCVR) amino acidsequence selected from the group consisting of SEQ ID NO:10, 26, 42, 58,74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298,314, 322, 368, and 384, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises a HCVR amino acid sequence and a LCVR amino acidsequence, wherein the HCVR/LCVR sequence pair is selected from the groupconsisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106,114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234,242/250, 258/266, 274/282, 290/298, 306/314, 114/322, 360/368, and376/384.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises a HCVR amino acid sequence and a LCVR amino acidsequence, wherein the HCVR/LCVR sequence pair is selected from the groupconsisting of (HCVR/LCVR): 21-E5 (SEQ ID NO:34/42); 21-B9 (SEQ IDNO:18/26); 21-E9 (SEQ ID NO:98/106); 21-A2 (SEQ ID NO:2/10); 22-D3 (SEQID NO:50/58); 22-E6 (SEQ ID NO:66/74); 22-G10 (SEQ ID NO:82/90); 1A2(SEQ ID NO:226/234); 20B12 (SEQ ID NO:274/282); 58C8 (SEQ IDNO:242/250); 19F2 (SEQ ID NO:258/266); 8D12-1 (SEQ ID NO:114/122); 4E3-7(SEQ ID NO:194/202); 9B11-12 (SEQ ID NO:162/170); 4B9 (SEQ IDNO:226/234); 1H4-5 (SEQ ID NO:210/218); 9B4-3 (SEQ ID NO:178/186); 3E2-1(SEQ ID NO:290/298); 4G3-25 (SEQ ID NO:306/314); 4B6-6 (SEQ IDNO:130/138); H4H1657N2 (SEQ ID NO:360/368); H4H1669P (SEQ IDNO:376/384).

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises a heavy chain complementarity determining region 3(HCDR3) domain and a light chain CDR3 (LCDR3) domain, wherein the HCDR3domain has an amino acid sequence selected from the group consisting ofSEQ ID NO:8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216,232, 248, 264, 280, 296, 312, 366, and 382, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity, and the LCDR3 domain has an amino acidsequence selected from the group consisting of SEQ ID NO:16, 32, 48, 64,80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304,320, 328, 374, and 390, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity. In another embodiment, an anti-GDF8 antibody orantigen-binding fragment thereof comprises an HCDR3/LCDR3 amino acidsequence pair selected from the group consisting of SEQ ID NO:8/16,24/32, 40/48, 56/64, 72/80, 88/96, 104/112, 120/128, 136/144, 152/160,168/176, 184/192, 200/208, 216/224, 232/240, 248/256, 264/272, 280/288,296/304, 312/320, 120/328, 366/374, and 382/390.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises heavy chain CDR1 (HCDR1) and CDR2 (HCDR2) domains andlight chain CDR1 (LCDR1) and CDR2 (LCDR2) domains, wherein the HCDR1domain has an amino acid sequence selected from the group consisting ofSEQ ID NO:4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212,228, 244, 260, 276, 292, 308, 362, and 378, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; the HCDR2 domain has an amino acid sequenceselected from the group consisting of SEQ ID NO:6, 22, 38, 54, 70, 86,102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310,364, and 380, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;the LCDR1 domain has an amino acid sequence selected from the groupconsisting of SEQ ID NO:12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172,188, 204, 220, 236, 252, 268, 284, 300, 316, 324, 370, and 386, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity and the LCDR2 domainhas an amino acid sequence selected from the group consisting of SEQ IDNO:14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238,254, 270, 286, 302, 318, 326, 372, and 388, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity. In another embodiment, the HCDR1, HCDR2 andHCDR3 domains have respective amino acid sequence combinations selectedfrom the group consisting of SEQ ID NO:36/38/40, 116/118/120,228/230/232, 362/364/366, and 378/380/382; and the LCDR1, LCDR2 andLCDR3 domains have respective amino acid sequence combinations selectedfrom the group consisting of SEQ ID NO:44/46/48, 124/126/128,236/238/240, 370/372/374, and 386/388/390.

In yet another embodiment, the heavy and light chain CDR domains of theanti-GDF8 antibody or antigen-binding fragment thereof(HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3) have amino acid sequencecombinations selected from the group consisting of SEQ ID NO:36/38/40/44/46/48 (e.g., 21-E5), 116/118/120/124/126/128 (e.g., 8D12),228/230/232/236/238/240 (e.g., 1A2), 362/364/366/370/372/374 (e.g.,H4H1657N2), and 378/380/382/386/388/390 (e.g., H4H1669P).

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises heavy and light chain CDR domains contained withinheavy and light chain variable region (HCVR/LCVR) amino acid sequencepairs selected from the group consisting of SEQ ID NO: 2/10, 18/26,34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298,306/314, 114/322, 360/368, and 376/384.

Methods and techniques for identifying CDRs within HCVR and LCVR aminoacid sequences are well known in the art and can be used to identifyCDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

In one embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises a HCVR having an amino acid sequence encoded by anucleic acid sequence selected from the group consisting of SEQ ID NO:1,17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241,257, 273, 289, 305, 359, and 375, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises a LCVR having an amino acid sequence encoded by anucleic acid sequence selected from the group consisting of SEQ ID NO:9,25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249,265, 281, 297, 313, 321, 367, and 383, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises a HCVR/LCVR pair having amino acid sequences encodedby a nucleic acid molecule pair selected from the group consisting ofSEQ ID NO: 1/9, 17/25, 33/41, 49/57, 65/73, 81/89, 97/105, 113/121,129/137, 145/153, 161/169, 177/185, 193/201, 209/217, 225/233, 241/249,257/265, 273/281, 289/297, 305/313, 113/321, 359/367, and 375/383.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises a HCDR3 domain having an amino acid sequence encodedby a nucleic acid sequence selected from the group consisting of SEQ IDNO:7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231,247, 263, 279, 295, 311, 365, and 381, ora substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity, and a LCDR3 domain having an amino acidsequence encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175,191, 207, 223, 239, 255, 271, 287, 303, 319, 327, 373, and 389, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity. In one embodiment,the HCDR3/LCDR3 domain pair has amino acid sequences encoded by anucleic acid sequence pair selected from the group consisting of SEQ IDNO:7/15, 23/31, 39/47, 55/63, 71/79, 87/95, 103/111, 119/127, 135/143,151/159, 167/175, 183/191, 199/207, 215/223, 231/239, 247/255, 263/271,279/287, 295/303, 311/319, 119/327, 365/373, and 381/389.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises HCDR1 and HCDR2 domains, and LCDR1 and LCDR2 domains,wherein the HCDR1 domain has an amino acid sequence encoded by a nucleicacid sequence selected from the group consisting of SEQ ID NO:3, 19, 35,51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275,291, 307, 361, and 377, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity, the HCDR2 domain has an amino acid sequence encoded by anucleic acid sequence selected from the group consisting of SEQ ID NO:5,21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245,261, 277, 293, 309, 363, and 379, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity, the LCDR1 domain has an amino acid sequence encodedby a nucleic acid sequence selected from the group consisting of SEQ IDNO:11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235,251, 267, 283, 299, 315, 323, 369, and 385, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity, and the LCDR2 domain has an amino acidsequence encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173,189, 205, 221, 237, 253, 269, 285, 301, 317, 325, 371, and 387.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof comprises heavy and light chain CDR domains(HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3) having amino acid sequencecombinations encoded by a nucleic acid sequence set of SEQ IDNO:35/37/39/43/45/47, 115/117/119/123/125/127, 227/229/231/235/237/239,361/363/365/369/371/373, or 377/379/381/385/387/389.

In a preferred embodiment, the anti-GDF8 antibody or antigen-bindingfragment thereof that specifically binds GDF8 comprises the HCDRs of aheavy chain variable region (HCVR) comprising SEQ ID NO:360 and theLCDRs of a light chain variable region (LCVR) comprising SEQ ID NO:368.In another embodiment, the anti-GDF8 antibody or antigen-bindingfragment that specifically binds GDF8 comprises three HCDRs comprisingSEQ ID NO:362, SEQ ID NO:364, and SEQ ID NO:366 and three LCDRscomprising SEQ ID NO:370, SEQ ID NO:372, and SEQ ID NO:374.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof is a fully human or humanized antibody or antibody fragment thatbinds GDF8 with an affinity (expressed as a dissociation constant, “KD”)of about 1 nM or less, as measured by surface plasmon resonance assay(for example, BIACORE™). In certain embodiments, the antibody of theinvention exhibits a KD of about 700 pM or less; about 500 pM or less;about 320 pM or less; about 160 pM or less; about 100 pM or less; about50 pM or less; about 10 pM or less; or about 5 pM or less.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof is a fully human or humanized monoclonal antibody (mAb) thatspecifically binds and inhibits human GDF8 and exhibits an IC50 of lessthan or equal to about 10 nM; about 5 nM or less; about 3 nM or less;about 2 nM or less; about 1 nM or less; about 500 pM or less; or about200 pM or less, as measured by GDF8 inducible luciferase assay.

In one embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof has a modified glycosylation pattern. In some applications,modification to remove undesirable glycosylation sites may be useful, oran antibody lacking a fucose moiety present on the oligosaccharidechain, for example, to increase antibody dependent cellular cytotoxicity(ADCC) function (Shield et al. (2002) JBC 277:26733). In otherapplications, modification of a galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof competes for specific binding to GDF8 with another antibodycomprising a HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 domain combinationhaving amino acid sequences selected from the group consisting of SEQ IDNO:36/38/40/44/46/48, 116/118/120/124/126/128, 228/230/232/236/238/240,362/364/366/370/372/374, or 378/380/382/386/388/390. In anotherembodiment, an anti-GDF8 antibody or antigen-binding fragment thereofcompetes for specific binding to GDF8 with another antibody comprising aHCVR/LCVR amino acid sequence pair of SEQ ID NO:2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298,306/314, 114/322, 360/368, or 376/384.

In another embodiment, an anti-GDF8 antibody or antigen-binding fragmentthereof recognizes the epitope on GDF8 that is recognized by anotherantibody comprising a HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 domaincombination having amino acid sequences selected from the groupconsisting of SEQ ID NO: 36/38/40/44/46/48, 116/118/120/124/126/128,228/230/232/236/238/240, 362/364/366/370/372/374, or378/380/382/386/388/390. In another embodiment, an anti-GDF8 antibody orantigen-binding fragment thereof recognizes the epitope on GDF8 that isrecognized by another antibody comprising a HCVR/LCVR amino acidsequence pair of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90,98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218,226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 114/322, 360/368,or 376/384.

Activin A Inhibitors

The present invention includes methods for altering body composition,inducing a reduction in fat mass, and/or increasing lean mass in asubject, and methods for treating a disease or disorder characterized byincreased fat mass in a subject comprising administering a compositioncomprising an effective amount of an Activin A inhibitor to the subject.

As used herein, an “Activin A inhibitor” is any agent that binds to orinteracts with human Activin A and interferes with or inhibits thenormal biological function of Activin A in vitro or in vivo.Non-limiting examples of categories of Activin A inhibitors includesmall molecule Activin A antagonists, nucleic acid-based inhibitors ofActivin A expression or activity (e.g., siRNA or antisense),peptide-based molecules that specifically interact with Activin A (e.g.,peptibodies), receptor molecules that specifically interact with ActivinA, Activin A-binding scaffold molecules, proteins comprising aligand-binding portion of a receptor that specifically binds Activin A,and anti-Activin A aptamers or portions thereof. In a preferredembodiment, an Activin A inhibitor that can be used in the context ofthe present invention is an anti-Activin A antibody or antigen-bindingfragment thereof that specifically binds human Activin A. Anti-Activin Aantibodies include neutralizing and/or blocking antibodies. Theinhibition caused by anti-Activin A neutralizing and/or blockingantibodies need not be complete, as long as it is detectable usingappropriate assays.

Activins are homo- and hetero-dimeric molecules comprising βA and/or βBsubunits. The βA subunit has the amino acid sequence of SEQ ID NO:617and the βB subunit has the amino acid sequence of SEQ ID NO: 619.Activin A is a homodimer of two βA subunits; Activin B is a homodimer oftwo βB subunits; and Activin AB is a heterodimer of one βA subunit andone βB subunit. An anti-Activin A antibody or antigen-binding fragmentthereof specifically binds the βA subunit. Since the βA subunit is foundin both Activin A and Activin AB molecules, an “anti-Activin A antibodyor antigen-binding fragment thereof” can specifically bind Activin A, aswell as Activin AB (by virtue of its interaction with the βA subunit).Therefore, an anti-Activin A antibody or antigen-binding fragmentthereof specifically binds Activin A, or Activin A and Activin AB, butdoes not bind other ActRIIB ligands, such as Activin B, GDF3, GDF8,BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, Nodal, etc.

In some embodiments, an anti-Activin A antibody or antigen-bindingfragment thereof is employed as set forth in U.S. Pat. No. 9,718,881.Exemplary anti-Activin A antibodies that can be used in the compositionsand methods of the invention include, e.g., the fully-human anti-Activinantibody H4H10446P2, also known as REGN2477, (e.g., an anti-Activin Aantibody comprising the heavy and light chain variable regions havingamino acid sequences SEQ ID NO: 162 and SEQ ID NO: 146, respectively, asset forth in U.S. Pat. No. 9,718,881).

Table 2 sets forth heavy and light chain variable region amino acidsequence pairs of selected anti-Activin A antibodies and theircorresponding antibody identifiers that can be used in the compositionsand methods of the invention. The corresponding nucleic acid sequenceidentifiers are set forth in Table 3.

TABLE 2 anti-Activin A Amino Acid Sequence Identifiers SEQ ID NOs:Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3H4H10423P 393 395 397 399 401 403 405 407 H4H10424P 409 411 413 415 417419 421 423 H4H10426P 425 427 429 431 433 435 437 439 H4H10429P 441 443445 447 449 451 453 455 H4H10430P 457 459 461 463 465 467 469 471H4H10432P2 473 475 477 479 481 483 485 487 H4H10433P2 489 491 493 495481 483 485 487 H4H10436P2 497 499 501 503 481 483 485 487 H4H10437P2505 507 509 511 481 483 485 487 H4H10438P2 513 515 517 519 481 483 485487 H4H10440P2 521 523 525 527 481 483 485 487 H4H10442P2 529 531 533535 537 539 541 543 H4H10445P2 545 547 549 551 537 539 541 543H4H10446P2 553 555 557 559 537 539 541 543 H4H10447P2 561 563 565 567537 539 541 543 H4H10448P2 569 571 573 575 537 539 541 543 H4H10452P2577 579 581 583 537 539 541 543 H4H10468P2 585 −587 589 591 537 539 541543 H2aM10965N 593 595 597 599 601 603 605 607

TABLE 3 anti-Activin A Nucleic Acid Sequence Identifiers SEQ ID NOs:Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3H4H10423P 392 394 396 398 400 402 404 406 H4H10424P 408 410 412 414 416418 420 422 H4H10426P 424 426 428 430 432 434 436 438 H4H10429P 440 441444 446 448 450 452 454 H4H10430P 456 458 460 462 464 466 468 470H4H10432P2 472 474 476 478 480 482 484 486 H4H10433P2 488 490 492 494480 482 484 486 H4H10436P2 496 498 500 502 480 482 484 486 H4H10437P2504 506 508 510 480 482 484 486 H4H10438P2 512 514 516 518 480 482 484486 H4H10440P2 520 522 524 526 480 482 484 486 H4H10442P2 528 530 532534 536 538 540 542 H4H10445P2 544 546 548 550 536 538 540 542H4H10446P2 552 554 556 558 536 538 540 524 H4H10447P2 560 562 564 566536 538 540 542 H4H10448P2 568 570 572 574 536 538 540 542 H4H10452P2576 578 580 582 536 538 540 542 H4H10468P2 584 586 588 590 536 538 540542 H2aM10965N 592 594 596 598 600 602 604 606

In one embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a heavy chain variable region (HCVR) havingan amino acid sequence selected from the group consisting of SEQ ID NO:393, 409, 425, 441, 457, 473, 489, 497, 505, 513, 521, 529, 545, 553,561, 569, 577, 585, and 593, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a light chain variable region (LCVR) havingan amino acid sequence selected from the group consisting of SEQ ID NO:401, 417, 433, 449, 465, 481, 537, and 601, ora substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a HCVR and LCVR (HCVR/LCVR) amino sequencepair selected from the group consisting of SEQ ID NO: 393/401, 409/417,425/433, 441/449, 457/465, 473/481, 489/481, 497/481, 505/481, 513/481,521/481, 529/537, 545/537, 553/537, 561/537, 569/537, 577/537, 585/537,and 593/601.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a heavy chain CDR3 (HCDR3) domain having anamino acid sequence selected from the group consisting of SEQ ID NO:399, 415, 431, 447, 463, 479, 495, 503, 511, 519, 527, 535, 551, 559,567, 575, 583, 591, and 599, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a light chain CDR3 (LCDR3) domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 407, 423, 439,455, 471, 487, 543, and 607, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a HCDR3/LCDR3 amino acid sequence pairselected from the group consisting of SEQ ID NO: 399/407, 415/423,431/439, 447/455, 463/471, 479/487, 495/487, 503/487, 511/487, 519/487,527/487, 535/543, 551/543, 559/543, 567/543, 575/543, 583/543, 591/543,and 599/607.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a heavy chain CDR1 (HCDR1) domain having anamino acid sequence selected from the group consisting of SEQ ID NO:395, 411, 427, 443, 459, 475, 491, 499, 507, 515, 523, 531, 547, 555,563, 571, 579, 587, and 595, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a heavy chain CDR2 (HCDR2) domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 397, 413, 429,445, 461, 477, 493, 501, 509, 517, 525, 533, 549, 557, 565, 573, 581,589, and 597, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;a light chain CDR1 (LCDR1) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 403, 419, 435, 451, 467, 483,539, and 603, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;and a light chain CDR2 (LCDR2) domain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 405, 421, 437, 453,469, 485, 541, and 605, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, having the amino acid sequences selected from the groupconsisting of: SEQ ID NOs: 395-397-8-403-405-407;411-413-415-419-421-423; 36-429-431-435-437-439;443-445-447-451-453-455; 459-461-463-467-469-471;475-477-479-483-485-487; 491-493-495-483-485-487;499-501-503-483-485-487; 507-509-511-483-485-487;515-517-519-483-485-487; 523-525-527-483-485-487;531-533-535-539-541-543; 547-549-551-539-541-543;555-557-559-539-541-543 (H4H10446P2); 563-565-567-539-541-543;571-573-575-539-541-543; 579-581-583-539-541-543;587-589-591-539-541-543; and 595-597-599-603-605-607.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises the heavy and light chain CDR domainscontained within heavy and light chain variable region (HCVR/LCVR)sequence pairs selected from the group consisting of SEQ ID NO: 393/401,409/417, 425/433, 441/449, 457/465, 473/481, 489/481, 497/481, 505/481,513/481, 521/481, 529/537, 545/537, 553/537,561/537, 569/537,577/537,585/537, and 593/601.

In one embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a HCVR having an amino acid sequence encodedby a nucleic acid sequence selected from the group consisting of SEQ IDNO:392, 408, 424, 440, 456, 472, 488, 496, 504, 512, 520, 528, 544, 552,560, 568, 576, 584, and 592, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a LCVR having an amino acid sequence encodedby a nucleic acid sequence selected from the group consisting of SEQ IDNO: 400, 416, 432, 448, 464, 480, 536, and 600, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a HCVR and a LCVR, wherein the HCVR/LCVR pairhas amino acid sequences encoded by a nucleic acid sequence pairselected from the group consisting of SEQ ID NO: 392/400, 408/416,424/432, 440/448, 456/464, 472/480, 488/480, 496/480, 504/480, 512/480,520/480, 528/536, 544/536, 552/536, 560/536, 568/536, 576/536, 584/536,and 592/600.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises a HCDR3 domain having an amino acid sequenceencoded by a nucleic acid sequence selected from the group consisting ofSEQ ID NO:398, 414, 430, 446, 462, 478, 498, 502, 510, 518, 526, 534,550, 558, 566, 574, 582, 590, and 598, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity, and a LCDR3 domain having an amino acidsequence encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:406, 422, 435, 454, 470, 486, 542, and 606, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity. In one embodiment,the HCDR3/LCDR3 domain set has amino acid sequences encoded by a nucleicacid sequence pair selected from the group consisting of SEQ IDNO:398/406, 414/422, 430/438, 446/454, 462/470, 478/486, 494/486,502/486, 510/486, 518/486, 526/486, 534/542, 550/542, 558/542, 566/542,574/542, 582/542, 590/542, and 598/606.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises HCDR1 and HCDR2 domains, and LCDR1 and LCDR2domains, wherein the HCDR1 domain has an amino acid sequence encoded bya nucleic acid sequence selected from the group consisting of SEQ IDNO:394, 410, 426, 442, 458, 474, 490, 498, 506, 514, 522, 530, 546, 554,562, 570, 578, 586, and 594, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity, the HCDR2 domain has an amino acid sequence encoded by anucleic acid sequence selected from the group consisting of SEQ IDNO:396, 412, 428, 444, 460, 476, 492, 500, 508, 516, 524, 532, 548, 556,564, 572, 580, 588, and 596, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity, the LCDR1 domain has an amino acid sequence encoded by anucleic acid sequence selected from the group consisting of SEQ IDNO:402, 418, 434, 450, 466, 482, 538, and 602, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity, and the LCDR2 domain has an aminoacid sequence encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:404, 420, 436, 452, 468, 484, 540, and 604, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof comprises heavy and light chain CDR (HCDR1, HCDR2,HCDR3, LCDR1, LCDR2, LCDR3) domains having amino acid sequences encodedby a nucleic acid sequence set selected from the group consisting of SEQID NO: 394/396/398/402/404/406, 410/412/414/418/420/422,426/428/430/434/436/438, 442/444/446/450/452/454,458/460/462/466/468/470, 474/476/478/482/484/486,490/492/494/482/484/486, 498/500/502/482/484/486,506/508/510/482/484/486, 514/516/518/482/484/486,522/524/526/482/484/486, 530/532/534/538/540/542,546/548/550/538/540/542, 554/556/558/538/540/542,562/564/566/538/540/542, 570/572/574/538/540/542,578/580/582/538/540/542, 586/588/590/538/540/542, and594/596/598/602/604/606.

In one embodiment, the anti-Activin A antibody or antigen-bindingfragment thereof comprises the HCVR and LCVR (HCVR/LCVR) amino sequencepair of SEQ ID NO: 553/537 and the anti-GDF8 antibody or antigen-bindingfragment thereof comprises a HCVR and LCVR (HCVR/LCVR) amino sequencepair of SEQ ID NO: 360/368.

In another embodiment, the anti-Activin A antibody or antigen-bindingfragment thereof comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, having the amino acid sequences of: SEQ ID NOs:555-557-559-539-541-543 (H4H10446P2), and the anti-GDF8 antibody orantigen-binding fragment thereof comprisesHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences of: SEQ ID NOs: 362/364/366/370/372/374 (e.g.,H4H1657N2).

In another embodiment, an anti-Activin A antibody or antigen-bindingfragment thereof has a modified glycosylation pattern. In someapplications, modification to remove undesirable glycosylation sites maybe useful, or an antibody lacking a fucose moiety present on theoligosaccharide chain, for example, to increase antibody dependentcellular cytotoxicity (ADCC) function (Shield et al. (2002) JBC277:26733). In other applications, modification of a galactosylation canbe made in order to modify complement dependent cytotoxicity (CDC).

The fully-human anti-Activin A and/or anti-GDF8 antibodies describedherein may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. The compositionsand methods of the invention use, in additional embodiments, antibodiesand antigen-binding fragments thereof that are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are back-mutated to thecorresponding germline residue(s) or to a conservative amino acidsubstitution (natural or non-natural) of the corresponding germlineresidue(s) (such sequence changes are referred to herein as “germlineback-mutations”). A person of ordinary skill in the art, starting withthe heavy and light chain variable region sequences described herein,can easily produce numerous antibodies and antigen-binding fragmentsthat comprise one or more individual germline back-mutations orcombinations thereof. In certain embodiments, all of the frameworkand/or CDR residues within the VH and/or VL domains are mutated back tothe germline sequence. In other embodiments, only certain residues aremutated back to the germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. Furthermore, the antibodies and antigen-binding fragments used inthe compositions and methods of the invention may contain anycombination of two or more germline back-mutations within the frameworkand/or CDR regions, i.e., wherein certain individual residues aremutated back to the germline sequence while certain other residues thatdiffer from the germline sequence are maintained. Once obtained,antibodies and antigen-binding fragments that contain one or moregermline back-mutations can be easily tested for one or more desiredproperty such as, improved binding specificity, increased bindingaffinity, improved or enhanced antagonistic or agonistic biologicalproperties (as the case may be), reduced immunogenicity, etc. Antibodiesand antigen-binding fragments obtained in this general manner areencompassed within the invention.

The compositions and methods of the invention use, in additionalembodiments, anti-GDF8 antibodies and/or anti-Activin A antibodies (orantigen-binding fragments thereof) comprising variants of any of theHCVR, LCVR, and/or CDR amino acid sequences described herein having oneor more conservative substitutions. For example, anti-GDF8 antibodiesand/or anti-Activin A antibodies used in the compositions and methods ofthe invention have, in some embodiments, HCVR, LCVR, and/or CDR aminoacid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 orfewer, etc. conservative amino acid substitutions relative to any of theHCVR, LCVR, and/or CDR amino acid sequences described herein.

Bispecific Antibodies

Bispecific antibodies (bsAbs) combine specificities of two antibodiesand simultaneously bind different antigens or epitopes. Two or moreantigen-recognizing elements are engineered into a single antibody. Inone embodiment of the methods of the invention, the compositioncomprises an antibody comprising a GDF8-specific binding domain and anActivin A-specific binding domain. The term (antigen)“-specific bindingdomain,” as used herein, includes polypeptides comprising or consistingof: (i) an antigen-binding fragment of an antibody molecule, (ii) apeptide that specifically interacts with a particular antigen (e.g., apeptibody), and/or (iii) a ligand-binding portion of a receptor thatspecifically binds a particular antigen. For example, included arebispecific antibodies with one arm comprising a first heavy chainvariable region/light chain variable region (HCVR/LCVR) pair thatspecifically binds GDF8 and another arm comprising a second HCVR/LCVRpair that specifically binds Activin A.

Bispecific antibodies can be prepared according to known methods,including chemical cross-linking, hybrid hybridomas/quadromas, knobsinto holes, CrossMab, dual-variable-domain immunoglobulin, recombinantengineering (tandem single chain variable fragments/diabodies), and dockand lock. Other exemplary bispecific formats that can be used in thecontext of the present invention include, without limitation, e.g.,IgG-scFv fusions, dual variable domain (DVD)-Ig, common light chain,CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual actingFab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al.,mAbs 4:6, 1-11 (2012), and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates, which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J Am Chem Soc. 135(1):340-346 (2013)).

Specific Binding

The term “specifically binds” or the like, as used herein, means that anantigen-specific binding protein, or an antigen-specific binding domain,forms a complex with a particular antigen characterized by adissociation constant (K_(D)) of 500 pM or less, and does not bind otherunrelated antigens under ordinary test conditions. “Unrelated antigens”are proteins, peptides or polypeptides that have less than 95% aminoacid identity to one another. Methods for determining whether twomolecules specifically bind one another are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. For example, an antigen-specific binding protein or anantigen-specific binding domain, as used in the context of the presentinvention, includes antibodies or antigen-binding fragments thereof thatbind a particular antigen (e.g., GDF8, Activin A) or a portion thereofwith a K_(D) of less than about 500 pM, less than about 400 pM, lessthan about 300 pM, less than about 200 pM, less than about 100 pM, lessthan about 90 pM, less than about 80 pM, less than about 70 pM, lessthan about 60 pM, less than about 50 pM, less than about 40 pM, lessthan about 30 pM, less than about 20 pM, less than about 10 pM, lessthan about 5 pM, less than about 4 pM, less than about 2 pM, less thanabout 1 pM, less than about 0.5 pM, less than about 0.2 pM, less thanabout 0.1 pM, or less than about 0.05 pM, as measured in a surfaceplasmon resonance assay.

Antibody binding (of antigen) can be quantitated in terms of K_(D), ameasurement of affinity. The lower the K_(D) value, the higher thebinding affinity of the antibody. The term “K_(D)”, as used herein, isintended to refer to the equilibrium dissociation constant of aparticular antibody-antigen interaction. Surface plasmon resonance canbe used to measure ligand binding, for example, antibody-antigeninteraction.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. For example, an antibody is said tospecifically bind an antigen when the K_(D) is less than or equal to10⁻⁸ M, less than or equal to 10⁻⁹ M, or less than or equal to 10⁻¹⁰ M.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the invention to make human antibodies thatspecifically bind to GDF8 and/or to Activin A.

Using VELOCIMMUNE™ technology or any other known method for generatingmonoclonal antibodies, high affinity chimeric antibodies to GDF8 and/orActivin A are initially isolated having a human variable region and amouse constant region. As in the experimental section below, theantibodies are characterized and selected for desirable characteristics,including affinity, selectivity, epitope, etc. The mouse constantregions are replaced with a desired human constant region to generatethe fully human antibody of the invention, for example wild-type ormodified IgG1 or IgG4. While the constant region selected may varyaccording to specific use, high affinity antigen-binding and targetspecificity characteristics reside in the variable region.

In general, the antibodies used in the methods of the instant inventionpossess very high affinities, typically possessing K_(D) of from about10⁻¹² through about 10⁻⁹ M, when measured by binding to antigen eitherimmobilized on solid phase or in solution phase.

Pharmaceutical Compositions and Methods of Administration

The present invention includes methods for altering body composition ofa subject. As used herein, the phrase “altering body composition” refersto a change in one or more of lean mass, fat mass, and/or bone mass in asubject. In some embodiments, body composition in a subject may bealtered by administering an effective amount of a GDF8 inhibitor and anActivin A inhibitor to the subject. Lean mass may be, for example, thighmuscle volume, appendicular lean body mass, or total lean mass, etc. Insome aspects, the thigh muscle volume may refer to thigh muscle tissuevolume excluding intramuscular adipose tissue and large vessels. In someaspects, the thigh muscle volume may refer to thigh muscle tissue volumeincluding intramuscular adipose tissue and large vessels. In someaspects, the appendicular lean body mass may be calculated by, forexample, aLBM equation. In some aspects, the appendicular lean mass maybe calculated by the sum of lean mass of arms and legs. Fat mass may be,for example, total fat mass, android fat mass, sum of intramuscular andperimuscular adipose tissue (IMAT), subcutaneous adipose tissue volume,sum of fat mass of arms and legs, thigh intramuscular adipose tissue,etc. Bone mass may be, for example, total bone mineral density(BMD)mass, total bone mineral content (BMC) mass, etc. In some embodiments,alteration of body composition comprises an increase in muscle massand/or a reduction of fat mass. In some embodiments, alteration of bodycomposition comprises an increase in muscle mass and a reduction of fatmass simultaneously. In some embodiments, alteration of body compositioncomprises an increase in muscle mass and a reduction of fat masssimultaneously, without reduction in bone mass. In some embodiments,alteration of body composition comprises an increase in bone mineralcontent mass. In some embodiments, alteration of body compositioncomprises a decrease in total fat mass, android fat mass, and/orsubcutaneous fat mass. In some embodiments, alteration of bodycomposition comprises a decrease in total fat mass, android fat mass,and/or subcutaneous fat mass, without a reduction in thigh intramuscularadipose tissue volume.

The present invention includes methods for altering body composition,for example, inducing a reduction in fat mass in a subject and methodsfor treating a disease or disorder characterized by increased fat mass,comprising administering a first composition comprising an effectiveamount of a GDF8 inhibitor and a second composition comprising aneffective amount of an Activin A inhibitor to the subject. The first andsecond compositions can be administered concurrently or sequentially tothe subject. The first and second compositions can also be combined intoa third composition prior to administration. Thus, in certainembodiments, a composition comprising both a GDF8 inhibitor and anActivin A inhibitor can be administered to a subject. The GDF8 inhibitorin such a composition can, for example, be an anti-GDF8 antibody. TheActivin A inhibitor in such a composition can, for example, be ananti-Activin A antibody.

The pharmaceutical compositions of the invention are formulated withsuitable carriers, excipients, and other agents that provide suitabletransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Powell et al. “Compendium of excipients forparenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

Various delivery systems are known and can be used to administer thepharmaceutical compositions of the present invention, e.g.,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the mutant viruses, receptor mediatedendocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).Methods of administration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical compositions of the presentinvention can be delivered in a controlled release system. In oneembodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRCCrit. Ref. Biomed. Eng. 14:201). In another embodiment, polymericmaterials can be used; see, Medical Applications of Controlled Release,Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, 1984, in Medical Applications of ControlledRelease, supra, vol. 2, pp. 115-138). Other controlled release systemsare discussed in the review by Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by knownmethods. For example, the injectable preparations may be prepared, e.g.,by dissolving, suspending or emulsifying the antibody or its saltdescribed above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc.

Dosage

The amount of active ingredient (e.g., anti-GDF8 antibody and/oranti-Activin A antibody) that can be administered to a subject is,generally, a therapeutically effective amount. The term “effectiveamount” is a concentration or amount of an active ingredient, forexample, an antibody or antigen-binding fragment of an antibody, whichresults in achieving a particular stated purpose. The term “effectiveamount” is used interchangeably with the term “therapeutically effectiveamount” and signifies a concentration or amount of an active ingredient,for example, an antibody or antigen-binding fragment thereof, which iseffective for achieving a stated therapeutic effect. The(therapeutically) effective amount may be determined empirically.

As used herein, the phrase “therapeutically effective amount” or“effective amount” means a dose of antigen-specific binding proteinsand/or antigen-binding molecules (e.g., antibodies) that results in adetectable decrease in fat mass. The effective amount may also, incertain embodiments, result in an increase in one or more of thefollowing parameters: body weight, muscle mass (e.g., tibialis anterior[TA] muscle mass, gastrocnemius [GA] muscle mass, quadriceps [Quad]muscle mass, appendicular lean body mass, etc.), muscle volume (e.g.,thigh muscle volume), muscle strength/power, and/or muscle function, andglucose tolerance.

A “therapeutically effective amount” or “effective amount” of a GDF8inhibitor (e.g., anti-GDF8 antibody) and/or an Activin A inhibitor(e.g., anti-Activin A antibody) includes, e.g., an amount of GDF8inhibitor and/or Activin A inhibitor that, when administered to asubject, causes a decrease in total fat mass of at least about 2% to 8%,at least 2.5% to 6%, at least 3% to 4%, or at least about 2.0%, at leastabout 2.5%, at least about 3.0%, or at least about 3.5%, or more. Forexample, a “therapeutically effective amount” or “effective amount” of aGDF8 inhibitor (e.g., anti-GDF8 antibody) and/or an Activin A inhibitor(e.g., anti-Activin A antibody) includes, e.g., an amount of GDF8inhibitor and/or Activin A inhibitor that, when administered to asubject, causes a decrease in total fat mass of at least about 3.5% ormore.

In some embodiments, a “therapeutically effective amount” or “effectiveamount” of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and/or an ActivinA inhibitor (e.g., anti-Activin A antibody) includes, e.g., an amount ofGDF8 inhibitor and/or Activin A inhibitor that, when administered to asubject, causes a decrease in android fat mass of at least about 2% to8%, at least 2.5% to 6%, at least 3% to 4%, or at least about 2.0%, atleast about 2.5%, at least about 3.0%, or at least about 3.5%, or more.For example, a “therapeutically effective amount” or “effective amount”of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and/or an Activin Ainhibitor (e.g., anti-Activin A antibody) includes, e.g., an amount ofGDF8 inhibitor and/or Activin A inhibitor that, when administered to asubject, causes a decrease in android fat mass of at least about 3.5%.

In certain embodiments, the amount also results in an increase in TA orGA muscle mass of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,60% or more, compared to control treated subjects.

In some embodiments, a “therapeutically effective amount” or “effectiveamount” of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and/or an ActivinA inhibitor (e.g., anti-Activin A antibody) includes, e.g., an amount ofGDF8 inhibitor and/or Activin A inhibitor that, when administered to asubject, causes a increase in thigh muscle volume of at least about 2%to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%, at least3.0%, or at least 3.5%, or more

In some embodiments, a “therapeutically effective amount” or “effectiveamount” of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and/or an ActivinA inhibitor (e.g., anti-Activin A antibody) includes, e.g., an amount ofGDF8 inhibitor and/or Activin A inhibitor that, when administered to asubject, causes a increase in total lean body mass of at least about 2%to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%, at least3.0%, or at least 3.5%, or more

In some embodiments, a “therapeutically effective amount” or “effectiveamount” of a GDF8 inhibitor (e.g., anti-GDF8 antibody) and/or an ActivinA inhibitor (e.g., anti-Activin A antibody) includes, e.g., an amount ofGDF8 inhibitor and/or Activin A inhibitor that, when administered to asubject, causes a increase in appendicular lean body mass of at leastabout 2% to 8%, 2.5% to 6%, 3% to 4%, or at least 2.0%, at least 2.5%,at least 3.0%, or at least 3.5%, or more.

In certain embodiments, the amount also results in an increase in TA orGA muscle mass of at least 2%, 3%, 5%, 10%, 15%, 20%, 25% or more,compared to control treated subjects.

In certain embodiments, a (therapeutically) effective amount of ananti-GDF8 antibody, anti-Activin A antibody, or bispecific antibody thatspecifically binds GDF8 and Activin A can be from about 0.05 mg to about600 mg; e.g., about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg,about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg,about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg,about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg,about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg,about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg,about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg,about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg,about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg,about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg,about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg,about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg,of the respective antibody. The dose may vary depending upon the age andthe size of a subject to be administered, target disease, conditions,route of administration, and the like. Depending on the severity of thecondition, the frequency and the duration of the treatment can beadjusted.

The amount of antibody (e.g., anti-GDF8 antibody, anti-Activin Aantibody, or bispecific antibody that specifically binds GDF8 andActivin A) contained within the individual doses may be expressed interms of milligrams of antibody per kilogram of patient body weight(i.e., mg/kg). For example, the anti-GDF8 antibody, anti-Activin Aantibody, and/or anti-GDF8/anti-Activin A bispecific antibody in thefirst, second, or third composition administered per the methods of theinvention may be administered to a patient at a dose of about 0.0001 toabout 50 mg/kg of patient body weight (e.g. 0.5 mg/kg, 1.0 mg/kg, 1.5mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg,5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5 mg/kg, 11.0mg/kg, 11.5 mg/kg, etc.).

The effective amount of a GDF8 inhibitor (e.g., an anti-GDF8 antibody)may, in certain embodiments, comprises a dosing regimen selected fromthe group consisting of at least 0.1 mg/kg to about 10 gm/kg, 1 mg/kg toabout 1 gm/kg, and 10 mg/kg to 100 mg/kg. The effective amount of anActivin A inhibitor (e.g., an anti-Activin A antibody) may, in certainembodiments, comprise a dosing regimen selected from the groupconsisting of at least 0.1 mg/kg to about 10 gm/kg, 1 mg/kg to about 1gm/kg, and 10 mg/kg to 100 mg/kg.

The effective amount of a GDF8 inhibitor (e.g., an anti-GDF8 antibody)may, in additional embodiments, comprise a dosing regimen selected froma group consisting of a single dose of about 0.01 to about 20 mg/kg bodyweight, about 0.1 to about 10 mg/kg body weight, and about 0.1 to about5 mg/kg body weight. The effective amount of an Activin A inhibitor(e.g., an anti-Activin A antibody) may, in additional embodiments,comprise a dosing regimen selected from a group consisting of a singledose of about 0.01 to about 20 mg/kg body weight, about 0.1 to about 10mg/kg body weight, and about 0.1 to about 5 mg/kg body weight. In onespecific aspect, the effective amount of an anti-GDF8 antibody is fromabout 2 mg/kg-10 mg/kg, 4 mg/kg-8 mg/kg, or about 6 mg/kg body weight ofsubject, and the effective amount of the anti-Activin A antibody is from0.5 mg/kg-15 mg/kg, 2 mg/kg-12 mg/kg, about 3 mg/kg, or about 10 mg/kgbody weight of a subject.

The first, second, and third compositions administered per methods ofthe invention may, in certain embodiments, comprise equal amounts ofGDF8 inhibitor (e.g., anti-GDF8 antibody) and/or Activin A inhibitor(e.g., anti-Activin A antibody). Alternatively, the amount of GDF8inhibitor (e.g., anti-GDF8 antibody) in the composition may be less thanor greater than the amount of Activin A inhibitor (e.g., an anti-ActivinA antibody). The effective amount of a GDF8 inhibitor (e.g., anti-GDF8antibody) may be lower, when in combination with an Activin A inhibitor(e.g., an anti-Activin A antibody), than in a separate composition. Theeffective amount of an Activin A inhibitor (e.g., an anti-Activin Aantibody) may be lower, when in combination with a GDF8 inhibitor (e.g.,anti-GDF8 antibody), than in a separate composition. A person ofordinary skill in the art, using routine experimentation, will be ableto determine the appropriate amounts of the individual components in thecompositions necessary to produce a desired therapeutic effect.

Aspects of the Disclosure

The disclosure provides compositions, kits, and methods of using GDF8inhibitors and Activin A inhibitors to reduce fat mass (induce areduction in fat mass) in a subject. The disclosure also providescompositions, kits, and methods of using GDF8 inhibitors and Activin Ainhibitors to treat diseases, disorders, and/or conditions associatedwith or characterized by increased fat mass in a subject. In preferredembodiments, the GDF8 inhibitor is an antibody or antigen-bindingfragment thereof that specifically binds GDF8.

Therapeutic Methods

The present invention includes methods for altering body composition,for example, methods for inducing a reduction in fat mass in a subject,methods for increasing muscle mass in a subject, and methods fortreating a disease or disorder characterized by increased fat mass, byspecifically binding GDF8 and/or Activin A. For example, the presentinvention includes methods for inducing a reduction in fat mass in asubject, inducing an increase in muscle mass in a subject, and methodsfor treating a disease or disorder characterized by increased fat massin a subject, by administering to the subject i) a compositioncomprising an anti-GDF8 antibody and a composition comprising ananti-Activin A antibody or ii) a composition comprising both ananti-GDF8 antibody and an anti-Activin A antibody or iii) a compositioncomprising a bispecific antibody comprising a first variable domaincomprising a HCVR/LCVR pair that specifically binds GDF8 and a secondvariable domain comprising a HCVR/LCVR pair that specifically bindsActivin A. Any of the GDF8 inhibitors and/or Activin A inhibitorsdisclosed or referred to herein can be used in the context of theseaspects of the invention.

In methods comprising administering a GDF8 inhibitor and an Activin Ainhibitor to a subject, the GDF8 inhibitor (for example, an anti-GDF8antibody) and the Activin A inhibitor (for example, an anti-Activin Aantibody) may be administered to the subject at the same orsubstantially the same time, e.g., in a single therapeutic dosage (thirdcomposition) or in two separate dosages (first and second compositions),which are administered simultaneously or within less than about 5minutes of one another. Alternatively, the GDF8 inhibitor and theActivin A inhibitor (first and second compositions) may be administeredto the subject sequentially, e.g., in separate therapeutic dosagesseparated in time from one another by more than about 5 minutes.

The reduction of fat mass in the subject of the methods according to theinvention can be a reduction in total fat mass as measured by DXA(Dual-energy X-ray absorptiometry).

In another embodiment, the reduction of fat mass in the subject of themethods according to the invention is a reduction in android fat mass(i.e., visceral fat associated with the upper/central body) as measuredby DXA (Dual-energy X-ray absorptiometry). In android obesity, thesubject stores fat around his or her abdominal region. Android obesitycan also be manifested in other areas of the upper trunk like the upperchest (front or back) nape area of the neck, and even the shoulders.Subjects who are android obese are at greater risk for obesity-relateddiseases/disorders like heart disease, and metabolic syndrome. Thelikelihood of developing gout, arterial-related diseases (due to highblood pressure) and many kinds of cancers are also linked to the centraltype of fat distribution in subjects who exhibit android obesity.

Body fat assessments are varied in precision and accuracy. Commonanthropometric measures include: weight, waist circumference, andskinfold measurements using skin calipers. More complex methods include:bioelectrical impedance analysis (BIA), the BOD POD, and dual-energyX-ray absorptiometry (DEXA or DXA). DXA is especially accurate andvalid, because it considers bone mineral content when estimating bodyfat and muscle. DEXA scans can evaluate different areas of fatdistribution to determine the android/gynoid fat ratio, which isdistinct from body mass index. DXA can measure total fat mass, totalbody muscle mass, visceral fat (fat around the organs) levels,intramuscular fat (fat between the muscles), total bone mineral density,and can even provide regional breakdowns. Finally, DXA can accuratelyassess the distribution of body fat associated with increased insulinresistance.

Avoidance of Side Effects

The present invention includes methods for altering body composition,for example, inducing a reduction in fat mass in a subject, and methodsfor treating a disease or disorder characterized by increased fat mass,comprising administering a GDF8 inhibitor and an Activin A inhibitor tothe subject, without causing adverse side effects associated with theadministration of molecules which bind multiple (e.g., 3 or more)ActRIIB ligands, for example, as set forth in U.S. Pat. No. 8,871,209.For example, the clinical molecule referred to as ACE-031 (AcceleronPharma, Inc., Cambridge, Mass.) is a multimer consisting of theextracellular portion of ActRIIB fused to an IgG Fc domain (thismolecule is also referred to herein as “ActRIIB-Fc”). ActRIIB-Fc bindsGDF8 as well as other ActRIIB ligands such as, e.g., Activin A, ActivinB, GDF11, BMP9, BMP10, and TGFβ, and is known to cause various adverseside effects when administered to human patients. For example,administration of ACE-031 to postmenopausal women in a Phase Ibascending dose study was shown to cause undesired increases inhemoglobin and decreases in FSH levels. In addition, a Phase II study ofACE-031 in pediatric patients with muscular dystrophy was discontinueddue to adverse effects including nose and gum bleeding. Dilated bloodvessels are also observed in patients treated with ActRIIB-Fc. Effect ofACE-031 in boys with Duchenne muscular dystrophy (DMD) demonstratedtrends for increased lean body mass and reduced fat mass butnon-muscle-related adverse events contributed to a decision todiscontinue the study. (Campbell, et al. 2017 Muscle Nerve 55: 458-464).Specifically inhibiting GDF8 and Activin A (e.g., by administering ananti-GDF8 antibody and an anti-Activin A antibody), while not inhibitingother ActRIIB ligands such as Activin B, GDF11, BMP9, BMP10, and TGFβ,results in an increase in a reduction in fat mass, without causing theadverse side effects associated with non-specific Activin-binding agentssuch as ActRIIB-Fc.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of the compositions of the present invention (e.g., compositionscomprising a GDF8 inhibitor and/or an Activin A inhibitor, for example,an anti-GDF8 antibody and/or an anti-Activin A antibody, or a bispecificantibody against GDF8 and Activin A), may be administered to a subjectover a defined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of the composition(s) of the present invention. As used herein,“sequentially administering” means that each dose of the compositions ofthe present invention are administered to the subject at a differentpoint in time, e.g., on different days separated by a predeterminedinterval (e.g., hours, days, weeks or months). The present inventionincludes methods that comprise sequentially administering to the patientan initial dose of a first and/or a second composition; or a thirdcomposition; followed by one or more secondary doses of the first and/orsecond composition; or the third composition; and optionally followed byone or more tertiary doses of the first and/or second composition; orthe third composition.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the compositions of thepresent invention. Thus, the “initial dose” is the dose that isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses that areadministered after the initial dose; and the “tertiary doses” are thedoses that are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of activeingredient(s), e.g., anti-GDF8 antibody and/or anti-Activin A antibody,but will generally differ from one another in terms of frequency ofadministration. In certain embodiments, however, the amount of activeingredient(s) contained in the initial, secondary and/or tertiary doseswill vary from one another (e.g., adjusted up or down as appropriate)during the course of treatment.

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or more) days after the immediately preceding dose.The phrase “the immediately preceding dose,” as used herein, means, in asequence of multiple administrations, the dose(s) of the compositions ofthe present invention that are administered to a subject prior to theadministration of the very next dose in the sequence, with nointervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof the compositions of the present invention. For example, in certainembodiments, only a single secondary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) secondary doses are administered to the patient. Likewise, incertain embodiments, only a single tertiary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to29 days after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 1 to 60days after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician, depending on the needs of the individual patient followingclinical examination.

In one embodiment, a subject may be subjected to preliminary DXA, thenreceive a composition comprising an anti-GDF8 antibody and ananti-Activin A antibody (or a composition comprising an anti-GDF8antibody and a composition comprising an anti-Activin A antibody), thenbe subjected to follow-up DXA. If the fat mass is not measurably reducedin the follow-up DXA (in comparison with the preliminary DXA), thesubject may receive the composition(s) again. Subsequent dosage amountand frequency of administration can, in an additional embodiment, bevaried based on the results of the follow-up DXA.

Combination Therapies

The methods of the present invention, according to certain embodiments,comprise administering to the subject one or more additional therapeuticagents that may be advantageously combined with the compositioncomprising a GDF8 inhibitor and/or an Activin A inhibitor. As usedherein, the expression “in combination with” means that the additionaltherapeutic agent(s) is/are administered before, after, or concurrentlywith a pharmaceutical composition comprising a GDF8 inhibitor and/or anActivin A inhibitor. The term “in combination with” also includessequential or concomitant administration of a GDF8 inhibitor, an ActivinA inhibitor, or both and a second therapeutic agent. The term“therapeutic agent” is also meant to include a specific therapy.

The additional therapeutic agent may be, e.g., another GDF8antagonist/inhibitor, another Activin A antagonist/inhibitor, growthfactor inhibitors, immunosuppressants, metabolic inhibitors, enzymeinhibitors, and cytotoxic/cytostatic agents, an IL-1 antagonist(including, e.g., an IL-1 antagonist as set forth in U.S. Pat. No.6,927,044), an IL-6 antagonist, an IL-6R antagonist (including, e.g., ananti-IL-6R antibody as set forth in U.S. Pat. No. 7,582,298), an IL-13antagonist, a tumor necrosis factor (TNF) antagonist, an IL-8antagonist, an IL-9 antagonist, an IL-17 antagonist, an IL-5 antagonist,an IgE antagonist, a CD48 antagonist, an IL-31 antagonist (including,e.g., as set forth in U.S. Pat. No. 7,531,637), a thymic stromallymphopoietin (TSLP) antagonist (including, e.g., as set forth in US2011/027468), interferon-gamma (IFNγ) antibiotics, topicalcorticosteroids, tacrolimus, pimecrolimus, cyclosporine, azathioprine,methotrexate, cromolyn sodium, proteinase inhibitors, systemiccorticosteroids, systemic immunotherapy, anti-histamines, chemotherapy,light therapy, or combinations thereof.

In further embodiments, the invention features a composition, theadditional therapeutic agent is selected from the group consisting of(1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductaseinhibitors, such as cerivastatin, atorvastatin, simvastatin,pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, andthe like; (2) inhibitors of cholesterol uptake and/or bile acidre-absorption; (3) niacin, which increases lipoprotein catabolism; (4)fibrates or amphipathic carboxylic acids, which reduce low-densitylipoprotein (LDL) level, improve high-density lipoprotein (HDL) and TGlevels, and reduce the number of non-fatal heart attacks; and (5)activators of the LXR transcription factor that plays a role incholesterol elimination such as 22-hydroxycholesterol, or fixedcombinations such as ezetimibe plus simvastatin; a statin with a bileresin (e.g., cholestyramine, colestipol, colesevelam), a fixedcombination of niacin plus a statin (e.g., niacin with lovastatin); orwith other lipid lowering agents such as omega-3-fatty acid ethyl esters(for example, omacor).

In still further embodiments, the second therapeutic agent is selectedfrom one or more other inhibitors/antagonists of glucagon or aninhibitor/antagonist of the glucagon receptor, as well as inhibitors ofother molecules, such as inhibitors of ANGPTL8 (for example, ananti-ANGPTL8 antibody), as well as inhibitors of other molecules, suchas ANGPTL3 (for example, an anti-ANGPTL3 antibody), ANGPTL4, ANGPTLS,ANGPTL6, apolipoprotein C-III (also referred to as APOC3; see forexample, inhibitors of APOC3 described in U.S. Pat. Nos. 8,530,439,7,750,141, 7,598,227 and volanesorsen, also referred to asISIS-APOCIIIRx) and proprotein convertase subtilisin/kexin type 9(PCSK9), which are involved in lipid metabolism, in particular,cholesterol and/or triglyceride homeostasis. Inhibitors of thesemolecules include small molecules, antisense molecules and antibodiesthat specifically bind to these molecules and block their activity.

The additional therapeutic agent may, in further embodiments, beselected from the group consisting of analgesics, anti-inflammatoryagents, including non-steroidal anti-inflammatory drugs (NSAIDS), suchas Cox-2 inhibitors, and the like, so as to ameliorate and/or reduce thesymptoms accompanying the underlying condition, if needed.

The additional therapeutic agent(s) may be administered prior to,concurrent with, or after the administration of the first and/or second;or third composition(s) described herein. For purposes of the presentdisclosure, such administration regimens may be considered theadministration of an anti-GDF8 antibody and/or an anti-Activin Aantibody “in combination with” a second therapeutically activecomponent.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.), but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1. Anti-GDF8 Alone Increased Total Lean Mass up to 3% inClinical Studies: Sarcopenia Phase 2 Data

A randomized, double-blind, placebo-controlled interventional studyphase 2 clinical trial was performed in patients 70 years and olderincluding men and postmenopausal women having sarcopenia. Patients weretreated 12 weeks with subcutaneous anti-GDF8 antibody REGN1033(H4H1657N2) alone, at either 100 mg anti-GDF8 antibody, Q4W s.c. (n=62);300 mg anti-GDF8 antibody Q4W s.c.(n=64), or 300 mg anti-GDF8 antibodyQ2W s.c. (n=59), or placebo (n=65). As shown in FIG. 1 significantincrease in total lean body mass compared to placebo was exhibited inpatients after 12 weeks when using REGN1033 alone at each of the threedoses, as shown in FIG. 1 and Table 4.

TABLE 4 Total Lean Mass % Change to Week 12 using anti-GDF8 REGN1033alone Anti-GDF8 Placebo 100 Q4W 300 Q4W 300 Q2W N 65 62 64 59 Baseline43.6 kg 42.9 kg 42.7 42.8 Mean Difference vs. — 1.66% 1.78% 2.29%placebo P value — 0.0077 0.0043 0.0004

The effects on strength and function were varied. Anti-GDF8 wasgenerally safe and well-tolerated (reactions, if any, were mild). Table4 shows patients receiving REGN1033 in either 100 mg or 300 mg dosesexhibited significantly increased Total lean mass as % change fromplacebo to week 12 data. Anti-GDF8 antibody REGN1033 alone increasedtotal lean body mass up to 3% in the study. The 300 mg regimens alsoresulted in decreases in total and android fat mass.

Example 2. Combination of Anti-GDF8 and Anti-Activin A First In-HumanSingle Ascending Dose Study

A randomized, double-blind placebo-controlled, ascending dose study wasinitiated to assess the tolerability and effects on body composition ofa combination of an intravenous anti-GDF8 antibody and an anti-Activin Aantibody vs. the individual components.

The primary objective of the study was to assess the safety andtolerability of an anti-Activin A antibody (e.g., H4H10446P2=REGN2477)alone and combined with an anti-GDF8 antibody (e.g., H4H1657N2=REGN1033)in healthy postmenopausal women aged 45 to 70 years of age.

Secondary objectives of the study included: an assessment of the effectof REGN2477 alone, REGN1033 alone, and REGN2477+REGN1033 in combinationon thigh muscle volume as measured by Magnetic Resonance Imaging (MRI),and an assessment of the effects of REGN2477 alone, REGN1033 alone andREGN2477+REGN1033 in combination on total and regional body compositionas measured by dual-energy X-ray absorptiometry (DXA)

Study Design

This study was a randomized, double-blind, placebo-controlled, ascendingdose study to assess the safety, tolerability, and pharmacodynamics ofintravenous REGN2477 (anti-Activin A) alone and in combination withREGN1033 (anti-GDF8) in healthy postmenopausal women.

A total of 48 subjects were randomized to 1 of the following 4sequential ascending REGN2477 IV dose panels where 8 subjects wererandomized in a 6:2 ratio into each of the first 3 Panels (Panels A, B,and C) and 24 subjects were randomized in a 1:1:1:1 ratio (6 subjectseach) into Panel D.

-   Panel A: 4 subjects REGN1033 (6 mg/kg IV)+REGN2477 low dose (1    mg/kg IV) or 2 subjects placebo-   Panel B: 4 subjects REGN1033 (6 mg/kg IV)+REGN2477 medium dose (3    mg/kg IV) or 2 subjects placebo-   Panel C: 4 subjects REGN1033 (6 mg/kg IV)+REGN2477 high dose (10    mg/kg IV) or 2 subjects placebo-   Panel D: REGN1033(6 mg/kg IV+REGN2477 high dose (10 mg/kg IV)    placebo, REGN2477 (10 mg/kg IV), or REGN1033 (6 mg/kg IV)

Subjects received a single intravenous dose of one or both of anti-GDF8antibody REGN1033 and/or anti-Activin A antibody REGN2477. In theprimary analyses, the placebo and high dose combination groups werepooled across panels, yielding 12 subjects on placebo and 12 on the highdose combination, as shown in FIG. 2A. The dosing schedule shown in FIG.2A was used in studies shown in each of the subsequent FIGS. 2B to 17.

Subjects participated in a screening period of up to 28 days, followedby a baseline and treatment visit on day 1, and a follow-up period of113 days.

Efficacy and Safety Analysis

The full analysis set (FAS) includes all randomized subjects; it isbased on the treatment allocated (as randomized). Efficacy endpointswere analyzed using the FAS. The safety analysis set (SAF) includes allrandomized subjects who received any study drug; it is based on thetreatment received (as treated). Treatment compliance/administration andall clinical safety variables were analyzed using the SAF.

The efficacy variables included: Thigh muscle tissue volume, excludingand including intramuscular adipose tissue and large vessels as measuredby Magnetic Resonance Imaging (MRI);Total lean mass as measured by dualX-ray absorptiometry (DXA); Appendicular lean body mass (calculated by aLBM equation) as measured by DXA; and Total fat mass as measured by DXA.

The demographics and baseline characteristics of the subjects werebalanced across the treatment groups, as per the below Table 5:

TABLE 5 Baseline Characteristics were balanced across treatment groupsAnti-GDF8 + Anti-GDF8 + Anti-GDF8 + Anti-Activin A anti-activin Aanti-activin A anti-activin A Placebo Anti-GDF8 (10 mg/kg) IV (1 mg/kg)IV (3 mg/kg) IV (10 mg/kg) IV (N = 12) (N = 6) (N = 6) (N = 6) (N = 6)(N = 12) Age (Years) Median 54 56 61 60 60 55.5 Height (cm) Median 162.5164.2 162.5 166.5 165 163.5 Weight (kg) Median 68.35 70.5 69.6 61.3567.05 70.4 BMI (kg/m²) Median 26.2 26.55 26.65 22.9 25.55 26.6

Statistical Methods

The percent change and change of efficacy variables from baseline toeither week 4 or 8 in the full analysis set (FAS) were analyzed usinganalysis of covariance (ANCOVA) model with treatment group as fixedeffect, and the baseline value as continuous covariate. Least-squaresmeans at week 4 and week 8 for each treatment group with thecorresponding standard error, confidence interval, and the p value fortreatment comparisons were provided from this model. Placebo subjectswere pooled across panels. Missing efficacy data was not imputed. Noadjustment for multiple testing is applied in this study.

Results

A total of 48 subjects were randomized, administered study drug andcompleted the study. One subject in the REGN2477+REGN1033 high dosegroup had an interrupted infusion of study drug due to an adverse eventof ‘Infusion site swelling’.

Efficacy

The blockade of both Activin A and GDF8 in combination increased thighmuscle volume and decreased fat mass at week 8, as shown in FIGS. 2B and2C. The greatest effect was seen at the highest dose combination.

FIG. 2B shows a bar graph showing % change in thigh muscle volume by MRIat 8 weeks after a single dose in postmenopausal women. Numbers showchanges from placebo. *indicates nominal p<0.05 vs. placebo;****indicates nominal p<0.0001 vs. placebo. The single dose amounts asmg/kg of either anti-Activin A antibody and/or anti-GDF8 antibody areshown below the bar graph. Treatment with REGN2477+REGN1033 in mediumand high dose combinations resulted in significantly increased thighmuscle volume (p<0.05; p<0.0001, respectively) in a dose-related mannercompared with placebo. Subjects in high dose panel exhibited up to 7.73%as % change from baseline versus 0.88% with placebo at week 8.

FIG. 2C shows a bar graph showing % change in total fat mass by DXA at 8weeks after a single dose in postmenopausal women. Numbers show changesfrom placebo. *indicates nominal p<0.05 vs. placebo; ****indicatesnominal p<0.0001 vs. placebo. The single dose amounts as mg/kg of eitheranti-Activin A antibody and/or anti-GDF8 antibody are shown below thebar graph. Treatment with high dose REGN2477+REGN1033 combinationresulted in significantly decreased total fat mass (p<0.05) comparedwith placebo.

As shown in FIG. 3, anti-Activin A antibody REGN2477 combined withanti-GDF8 antibody REGN1033 led to dose-dependent increases in thighmuscle volume. The thigh muscle volume results, as measured via MRI,excluding intermuscular adipose tissue and large vessels, are likewisesummarized in Table 6, below. Treatment with mid- and high doseREGN2477+REGN1033 combination resulted in significantly increased thighmuscle volume (p<0.05) compared with placebo, as shown in Table 6.

TABLE 6 Thigh Muscle Volume measured by MRI* anti- activin anti-GDF8 +anti-GDF8 + anti-GDF8 + anti- A High anti-activin A anti-activin Aanti-activin A Placebo GDF8 Dose Low Dose Mid Dose High Dose Week N 12 66 6 6 12 4, % mean 0.4 3.3 0.3 4.9 4.6 5.9 change Week mean 0.9 4.6 2.93.4 6.2 7.8 8, % Δ LSM 3.73 1.97 2.63 5.31 6.85 change vs (1.819)(1.821) (1.824) (1.817) (1.484) placebo (SE) Nominal 0.0467 0.28460.1569 0.0056 <0.0001 p-value *excluding intermuscular adipose tissueand large vessels

As shown in Table 4, treatment with REGN2477+REGN1033 in medium and highdose combinations resulted in significantly increased thigh musclevolume in a dose-related manner compared with placebo.

FIG. 4 shows that increases in thigh muscle volume were consistentlyobserved in individual subjects following treatment with anti-ActivinA+anti-GDF8 in combination. Within each treatment group, different linesindicate different individuals.

The pattern of effects on appendicular lean mass (the sum of lean massof arms and legs) with anti-Activin A antibody REGN2477+anti-GDF8antibody REGN1033 in combination was similar to that seen on thighmuscle volume, as shown in FIG. 5. The appendicular lean mass results,as measured via DXA, are likewise summarized in Table 7. The mid andhigh dosing combinations significantly increased appendicular lean masscompared to placebo, as shown in Table 7.

TABLE 7 Appendicular Lean Mass by DXA Anti-GDF8 + Anti-GDF8 +Anti-GDF8 + Anti-activin A anti-activin A anti-activin A anti-activin APlacebo Anti-GDF8 High Dose Low Dose Mid Dose High Dose N 12 6 6 6 6 12Baseline, Mean 17.44 18.10 18.02 17.92 18.67 17.32 kg Week 4, % Mean−0.2 1.2 2.1 4.2 4.6 3.8 Change Week 8, % Mean 0.8 2.6 2.3 3.7 6.9 5.8Change ΔLSM 1.90 1.61 2.96 6.39 4.97 vs (1.266) (1.265) (1.264) (1.275)(1.031) Placebo (SE) Nominal 0.1418 0.2109 0.0242 <0.0001 <0.0001p-value

As shown in FIG. 6, blockade of both Activin A and GDF8 led toreductions in total fat mass, as assessed by DXA. The high dosecombination of anti-GDF8 antibody REGN1033 and anti-Activin A antibodyREGN2477 significantly reduced total fat mass by DXA compared to placeboat week 8 (*p<0.05). The total fat mass results, as measured via DXA,are likewise summarized in Table 8, below.

TABLE 8 Total Fat Mass by DXA anti-GDF8 + anti-GDF8 + anti-GDF8 +anti-GDF8 anti-activin A anti-activin A anti-activin A anti-activin ADose High High Low Mid High Week 8, N 6 6 6 6 12 % Change ΔLSM vs −2.11(1.96) −0.69 (1.93) −0.2 (1.93) −2.08 (1.93) −3.92 (1.58)* Placebo (SE)*p < 0.05

Blockade of Activin A and GDF8 was also found to be associated withdecreases in android fat mass, as assessed by DXA, as shown in FIG. 7.The high dose combination of anti-GDF8 antibody REGN1033 andanti-Activin A antibody REGN2477 significantly reduced android fat massby DXA compared to placebo at week 8 (*p<0.05).

Further efficacy results are shown in FIGS. 8 to 17 and summarized inthe tables, below. REGN2477+REGN1033 high, medium and low doses areshown in FIG. 2A.

Compared with placebo, REGN2477+REGN1033 medium (p<0.05) and high dosegroups (p<0.001) exhibited significantly increased thigh muscle volume,excluding Intramuscular adipose tissue and large vessels (FIG. 8). Thighmuscle volume increased in the REGN2477+REGN1033 high dose group by7.73% as compared with 0.88% in the placebo group (nominal p<0.001) at 8weeks. Compared with placebo, REGN2477+REGN1033 at the medium dose andREGN1033 alone also significantly increased thigh muscle volume.Increases in thigh muscle volume were consistently observed inindividual subjects treated with the combination in a dose responsivemanner. (data not shown).

Compared with placebo, REGN2477+REGN1033 high dose group exhibitedsignificantly increased total lean mass by DXA (p<0.05) (FIG. 9).

Appendicular lean body mass (calculated via aLBM equation), wassignificantly increased in each of the combination REGN2477+REGN1033treatment groups compared with placebo (low dose p<0.05, medium and highdose groups p<0.001)(FIG. 10). Appendicular lean body mass increased inthe REGN2477+REGN1033 medium dose group by 7.15% from baseline ascompared with 0.76% in the placebo group at 8 weeks. Similarly,appendicular lean body mass increased in the REGN2477+REGN1033 high dosegroup by 5.7% from baseline as compared with 0.76% in the placebo group.

Total fat mass was significantly decreased in the high doseREGN2477+REGN1033 treatment group; total fat mass was decreased:3.92%(high dose group) compared with placebo at 0.5%(nominalp<0.05)(FIG. 11).

Compared to placebo, thigh muscle volume including intramuscular adiposetissue and large vessels was significantly increased in each of themedium and high dose REGN2477+REGN1033 groups and REGN1033 group at 4weeks, and at 8 weeks (FIG. 12). Low dose REGN2477+REGN1033 alsoexhibited significant increase in thigh muscle volume includingintramuscular adipose tissue and large vessels compared to placebo at 4weeks.

Appendicular lean mass (sum of arms and legs) was significantlyincreased in each of combination REGN2477+REGN1033 treatment groups(p<0.05) at 4 and 8 weeks compared to placebo (FIG. 13).

Android fat mass in the high dose REGN2477+REGN1033 treatment group wasalso significantly reduced (FIG. 14). Android fat mass was reduced 6.6%in the high dose REGN2477+REGN1033 group as compared to no reduction inthe placebo.

Thigh intramuscular adipose tissue volume (cm3) was significantlyincreased in the high dose REGN2477+REGN1033 group (p<0.05) as comparedto placebo at 8 weeks (FIG. 15).

Reductions in adipose tissue in the high dose group were observed in thesum of Intramuscular and Perimuscular Adipose Tissue (IMAT)(FIG. 16) andin the low and medium dose group of subcutaneous adipose tissue. (FIG.17). In contrast, thigh intramuscular adipose tissue was increased inthe high dose group to 8% as compared with placebo with a reduction of4%. (FIG. 17).

REGN2477+REGN1033, in the high dose group, significantly increased allof the major measures of muscle volume and lean mass compared withplacebo, at both Week 4 and Week 8; the effects at Week 4 were generallyless pronounced than at Week 8 (Table 9). A summary of Percent Change inKey Body Composition Measures at Week 8 (Full Analysis Set, LS Means andSE presented) is shown in Table 9, below.

TABLE 9 Summary of Change in Body Composition at Week 8 Low Medium HighR2477 R2477 R2477 R1033 R2477 (1 mg/kg) (3 mg/kg) (10 mg/kg) Placebo (6mg/kg) (10 mg/kg) +R1033 +R1033 +R1033 Endpoint (n = 12) (n = 6) (n = 6)(n = 6) (n = 6) (n = 12) Thigh % Change 0.88 4.61 2.85 3.51 6.19 7.73Muscle from (1.05) (1.49) (1.49) (1.49) (1.48) (1.05) Volume baselineDifference 3.73 1.97 2.63 5.31 6.85 from (1.82)* (1.82) (1.82) (1.82)*(1.48)** placebo Total % Change 1.31 2.18 1.94 3.16 4.67 4.31 Lean from(0.64) (0.89) (0.90) (0.89) (0.89) (0.63) Mass by baseline DXADifference 0.88 0.63 1.85 3.37 3.00 from (1.10) (1.11) (1.10) (1.10)*(0.89)* placebo Appendicular % Change 0.76 2.65 2.37 3.72 7.15 5.72 Leanfrom (0.73) (1.03) (1.03) (1.03) (1.04) (0.73) Body baseline MassDifference 1.90 1.61 2.96 6.39 4.97 from (1.27) (1.26) (1.26)* (1.27)**(1.03)** placebo Total Fat % Change −0.65 −2.76 −1.34 −0.85 −2.73 −4.57Mass by from (1.12) (1.60) (1.58) (1.58) (1.58) (1.12) DXA baselineDifference −2.11 −0.69 −0.2 −2.08 −3.92 from (1.96) (1.93) (1.93) (1.93)(1.58)* placebo *p < 0.05; **p < 0.001

In Table 9, above, changes from baseline and differences from placeboare Least-Squares (LS) means based on the ANCOVA model with baseline asa covariate and treatment as a fixed factor. Standard errors (SE) andp-values also taken from the ANCOVA. Nominal p-values are reported.

Bone mineral density (BMD) mass and bone mineral content (BMC) mass weremeasured by DXA, as shown in Table 10. At the high dose, R2477+R1033increased Bone Mineral Content as measured by DXA, while total bonemineral density did not change (Table 10).

Sum of fat mass of arms and legs were measured as shown in Table 10. Atthe high dose, R2477+R1033 decreased sum of fat mass of arms and legs(p<0.05).

The data from the primary endpoint analysis is shown in Table 10, below.

TABLE 10 Primary analysis of efficacy endpoints R2477 R1033 + R1033 +R1033 + (10 mg/kg) R2477 R2477 R2477 Analysis Placebo R1033 IV (1 mg/kg)(3 mg/kg) (10 mg/kg) Parameter Visit (n = 12) (n = 6) (n = 6) IV (n = 6)IV (n = 6) IV (n = 12) Thigh LS Mean 0.37 3.27 0.27 4.96 4.61 5.92Muscle (SE)Week 4 (0.76) (1.07) (1.08) (1.08) (1.07) (0.76) TissueVolume (cm3), Excluding Intra- muscular Adipose Tissue and LargeVessels/% change LS Mean 2.9  −0.11  4.59 4.24 5.55 Diff (SE)/  (1.32)*(1.32)  (1.32)*  (1.31)*  (1.07)** week 4 LS Mean 0.88 4.61 2.85 3.516.19 7.73 (SE)/week 8 (1.05) (1.49) (1.49) (1.49) (1.48) (1.05) LS Mean3.73 1.97 2.63 5.31 6.85 Diff (SE)/  (1.82)* (1.82) (1.82)  (1.82)* (1.48)** week 8 Change LS Mean 1.64 13.95  −0.14  20.29  18.77  24.5 (SE)/week 4 (3.26) (4.61) (4.63) (4.63) (4.61) (3.26) LS Mean 12.32 −1.78  18.65  17.14  22.86  Diff  (5.65)* (5.66)  (5.67)*  (5.64)* (4.61)** (SE)/week 4 LS Mean 3.5  20.42  11.22  14.55  25.44  31.95 (SE)/week 8 (4.13) (5.85) (5.87) (5.88) (5.85) (4.14) LS Mean 16.91 7.71 11.05  21.94  28.45  Diff (SE)/  (7.17)* (7.18) (7.19)  (7.16)* (5.85)** week 8 Thigh LS Mean 0.21 3.32 −0.2  5   4.49 5.76 Muscle(SE)/week 4 (0.78) (1.1)  (1.11) (1.11) (1.1)  (0.78) Volume (cm3),Including Intra- muscular Adipose Tissue and Large Vessels/% change LSMean 3.1  −0.41  4.79 4.28 5.54 Diff (SE)/  (1.35)* (1.36)  (1.35)* (1.35)*  (1.1)** week 4 LS Mean 0.85 4.14 2.47 3.14 5.99 7.41 (SE)/week8 (1.06) (1.51) (1.52) (1.51) (1.5)  (1.06) LS Mean 3.29 1.62 2.29 5.146.57 Diff (SE)/ (1.84) (1.85) (1.85)  (1.84)*  (1.5)** week 8 change LSMean 0.99 14.76  −2.61  21.22  19.35  25.22  (SE)/week 4 (3.45) (4.89)(4.94) (4.91) (4.89) (3.45) LS Mean 13.77  −3.6  20.24  18.36  24.23 Diff  (5.99)* (6.02)  (6.01)*  (5.98)*  (4.88)** (SE)/week 4 LS Mean3.55 19.54  9.26 13.64  26.04  32.27  (SE)/week 8 (4.42) (6.25) (6.32)(6.29) (6.25) (4.42) LS Mean 15.99  5.72 10.09  22.49  28.72  Diff (7.66)* (7.7)  (7.69)  (7.65)*  (6.25)** (SE)/week 8 Appendicular LSMean −0.31  1.26 2.15 4.26 4.77 3.7  Lean Body (SE)/week 4 (0.74) (1.04)(1.04) (1.04) (1.05) (0.74) Mass (Calculated by aLBM Equation) (kg)/%change LS Mean 1.57 2.46 4.57 5.08 4.01 Diff (SE)/ (1.28) (1.28) (1.28)**  (1.29)**  (1.04)** week 4 LS Mean 0.76 2.65 2.37 3.72 7.155.72 (SE)/week 8 (0.73) (1.03) (1.03) (1.03) (1.04) (0.73) LS Mean 1.9 1.61 2.96 6.39 4.97 Diff (1.27) (1.26)  (1.26)*  (1.27)**  (1.03)**(SE)/week 8 Change LS Mean −0.05  0.23 0.35 0.73 0.84 0.63 (SE)/week 4(0.13) (0.18) (0.18) (0.18) (0.18) (0.13) LS Mean 0.28 0.4  0.78 0.890.68 Diff (SE)/ (0.22) (0.22)  (0.22)*  (0.22)**  (0.18)** week 4 LSMean 0.11 0.45 0.41 0.64 1.28 1.02 (SE) (0.13) (0.19) (0.19) (0.18)(0.19) (0.13) LS Mean 0.33 0.29 0.53 1.16 0.91 Diff (0.23) (0.23) (0.23)*  (0.23)**  (0.18)** (SE)/week 8 Total Lean LS Mean 0.16 0.711.53 3.42 3.52 2.84 Mass (kg)/ (SE)/week 4 (0.56) (0.79) (0.79) (0.79)(0.79) (0.56) % change LS Mean 0.55 1.37 3.26 3.36 2.69 Diff (0.97)(0.98)  (0.98)*  (0.98)*  (0.79)* (SE)/week 4 LS Mean 1.31 2.18 1.943.16 4.67 4.31 (SE)/week 8 (0.64) (0.89) (0.9) (0.89) (0.89) (0.63) LSMean 0.88 0.63 1.85 3.37 3   Diff (1.1)  (1.11) (1.1)  (1.1)*  (0.89)*(SE)/week 8 change LS Mean 0.08 0.25 0.61 1.42 1.44 1.14 (SE)/week 4(0.23) (0.33) (0.33) (0.33) (0.33) (0.23) LS Mean 0.17 0.54 1.34 1.361.07 Diff (0.41) (0.41)  (0.41)*  (0.41)*  (0.33)* (SE)/week 4 LS Mean0.53 0.86 0.78 1.27 1.92 1.8  (SE)/week 8 (0.27) (0.37) (0.37) (0.37)(0.37) (0.26) LS Mean 0.33 0.25 0.74 1.39 1.26 Diff (0.46) (0.46) (0.46) (0.46)*  (0.37)* (SE)/week 8 Appendicular LS Mean −0.35  1.2  2.02 4.064.51 3.54 Lean Mass (SE)/week 4 (0.74) (1)   (1)   (1)   (1)   (0.71)(Sum of Lean Mass of Arms and Legs) (kg)/% change LS Mean 1.56 2.37 4.424.87 3.89 Diff (1.25) (1.24)  (1.24)**  (1.26)**  (1.02)** (SE)/week 4LS Mean 0.95 2.51 2.22 3.53 6.74 5.48 (SE)/week 8 (0.72) (0.97) (0.97)(0.97) (0.98) (0.69) LS Mean 1.56 1.28 2.58 5.79 4.53 Diff (1.22) (1.21) (1.21)*  (1.23)**  (0.99)** (SE)/week 8 change LS Mean −0.06  0.23 0.350.73 0.83 0.64 (SE)/week 4 (0.14) (0.18) (0.18) (0.18) (0.19) (0.13) LSMean 0.28 0.41 0.79 0.89 0.69 Diff (0.23) (0.23)  (0.23)*  (0.23)** (0.19)** (SE)/week 4 LS Mean 0.17 0.44 0.4  0.64 1.26 1.02 (SE)/week 8(0.14) (0.18) (0.18) (0.18) (0.18) (0.13) LS Mean 0.27 0.23 0.48 1.090.86 Diff (0.23) (0.23)  (0.23)*  (0.23)**  (0.19)** (SE)/week 8 TotalFat LS Mean −0.11  −1.15  1.32 −0.76  −1.52  −2.58  Mass (SE)/week 4(0.81) (1.15) (1.14) (1.14) (1.14) (0.8)  (kg)/% change LS Mean −1.04 1.42 −0.65  −1.41  −2.47  Diff (1.41) (1.39) (1.39) (1.39)  (1.14)*(SE)/week 4 LS Mean −0.65  −2.76  −1.34  −0.85  −2.73  −4.57  (SE)/week8 (1.12) (1.6)  (1.58) (1.58) (1.58) (1.12) LS Mean −2.11  −0.69  −0.2 −2.08  −3.92  Diff (1.96) (1.93) (1.93) (1.93)  (1.58)* (SE)/week 8change LS Mean 0.07 −0.18  0.29 −0.29  −0.38  −0.6  (SE)/week 4 (0.21)(0.3)  (0.29) (0.29) (0.29) (0.21) LS Mean −0.25  0.23 −0.36  −0.45 −0.67  Diff (SE)/ (0.36) (0.36) (0.36) (0.36)  (0.29)* week 4 LS Mean−0.04  −0.54  −0.46  −0.34  −0.75  −1.16  (SE)/week 8 (0.28) (0.4) (0.4)  (0.4)  (0.4)  (0.28) LS Mean −0.5  −0.42  −0.3  −0.71  −1.12 Diff (0.49) (0.49) (0.49) (0.49) (0.4)* (SE)/week 8 Android LS Mean−0.21  −1.57  0.44 −2.47  −1.8  −3.94  Fat Mass (SE)/week 4 (1.3) (1.85) (1.84) (1.84) (1.83) (1.3)  (kg)/% change LS Mean −1.35  0.65−2.25  −1.59  −3.73  Diff (2.26) (2.25) (2.25) (2.25)  (1.83)* (SE)/week4 LS Mean −0.05  −2.35  −4.67  −1.71  −2.62  −6.65  (SE) (1.69) (2.4) (2.39) (2.39) (2.38) (1.69) LS Mean −2.31  −4.62  −1.66  −2.57  −6.61 Diff (2.93) (2.92) (2.93) (2.92)  (2.38)* (SE)/week 8 change LS Mean0.03 0.01 0   −0.05  −0.04  −0.07  (SE)/week 4 (0.03) (0.04) (0.04)(0.04) (0.04) (0.03) LS Mean −0.01  −0.02  −0.07  −0.06  −0.1  Diff(0.05) (0.05) (0.05) (0.05)  (0.04)* (SE)/week 4 LS Mean 0.03 −0.01 −0.09  −0.03  −0.07  −0.13  (SE)/week 8 (0.04) (0.05) (0.05) (0.05)(0.05) (0.04) LS Mean −0.04  −0.11  −0.06  −0.1  −0.16  Diff (0.06)(0.06) (0.06) (0.06)  (0.05)* (SE)/week 8 Thigh LS Mean −2.9  −2   −3.44  6.19 −2.99  8.25 Intra- (SE)/week 4 (3)   (4.24) (4.23) (4.28)(4.24) (2.98) muscular Adipose Tissue Volume (cm3)/% volume LS Mean 0.9 −0.54  9.09 −0.09  11.15  Diff (5.22) (5.17) (5.27) (5.17)  (4.23)*(SE)/week 4 LS Mean −4.19  −3.35  0.65 1.14 −3.88  7.26 (SE)/week 8(4.21) (5.96) (5.95) (6.02) (5.96) (4.19) LS Mean 0.84 4.84 5.33 0.3111.45  Diff (7.34) (7.26) (7.41) (7.27) (5.95) (SE)/week 8 change LSMean −0.09  −0.07  −0.2  0.14 −0.12  0.24 (SE)/week 4 (0.13) (0.18)(0.18) (0.18) (0.18) (0.13) LS Mean 0.02 −0.11  0.23 −0.03  0.33 Diff(0.22) (0.22) (0.22) (0.22) (0.18) (SE)/week 4 LS Mean −0.17  −0.13 −0.02  −0.08  −0.22  0.2  (SE)/week 8 (0.17) (0.24) (0.24) (0.25) (0.24)(0.17) LS Mean 0.04 0.15 0.09 −0.05  0.37 Diff (0.3)  (0.3)  (0.3) (0.3)  (0.24) (SE)/week 8 Sum of LS Mean 0.98 −3.09  1.63 −2.09  1.75−1.54  Intra- (SE)/week 4 (1.4)  (1.98) (1.99) (2.03) (1.99) (1.4) muscular and Peri- muscular Adipose Tissue (IMAT)/% change LS Mean−4.07  0.65 −3.07  0.77 −2.52  Diff (2.43) (2.43) (2.47) (2.43) (1.98)(SE)/week 4 LS Mean 0.94 −0.12  0.26 −0.5  −1.63  −2.76  (SE)/week 8(1.49) (2.12) (2.13) (2.17) (2.12) (1.49) LS Mean −1.06  −0.68  −1.44 −2.57  −3.7  Diff (2.6)  (2.59) (2.64) (2.59) (2.11) (SE)/week 8 ChangeLS Mean 1.05 −3.45  2.29 −3.2  1.93 −1.75  (SE)/week 4 (1.65) (2.34)(2.35) (2.4)  (2.35) (1.65) LS Mean −4.5  1.24 −4.25  0.88 −2.8  Diff(2.87) (2.87) (2.92) (2.87) (2.33) (SE)/week 4 LS Mean 0.69 0.28 0.48−1.73  −1.97  −3.04  (SE)/week 8 (1.8)  (2.56) (2.57) (2.62) (2.57)(1.81) LS Mean −0.4  −0.21  −2.42  −2.66  −3.73  Diff (3.14) (3.13)(3.19) (3.13) (2.55) (SE)/week 8 Sub- LS Mean −1.27  0.05 −0.26  −1.67 −1.98  −1.76  cutaneous (SE)/week 4 (1.02) (1.45) (1.47) (1.44) (1.44)(1.02) Adipose Tissue Volume (cm3)/% change LS Mean 1.32 1.01 −0.4 −0.71  −0.49  Diff (1.78) (1.82) (1.76) (1.76) (1.44) (SE)/week 4 LSMean 1.31 1.01 1.56 −3.73  −3.04  −1.45  (SE)/week 8 (1.21) (1.71)(1.75) (1.71) (1.7)  (1.21) LS Mean −0.31  0.25 −5.04  −4.36  −2.77 Diff (2.11) (2.15)  (2.09)*  (2.09)* (1.7)  (SE)/week 8 change LS Mean−3.94  0.35 −0.74  −8.23  −3.02  −7.29  (SE)/week 4 (3.83) (5.42) (5.52)(5.39) (5.39) (3.82) LS Mean 4.29 3.19 −4.3  0.92 −3.35  Diff (6.68)(6.8)  (6.6)  (6.6)  (5.39) (SE)/week 4 LS Mean 4.74 5.7  5.53 −17.58 −8.75  −5.55  (SE)/week 8 (4.74) (6.7)  (6.83) (6.67) (6.66) (4.73) LSMean 0.95 0.79 −22.33  −13.5   −10.3   Diff (8.26) (8.41)  (8.16)*(8.17) (6.66) (SE)/week 8 Sum of Fat LS Mean −0.8  −0.59  1.89 0.48−2.87  −3.31  Mass of (SE)/week 4 (1.01) (1.44) (1.42) (1.42) (1.42)(1)   Arms and Legs (kg)/ % change LS Mean 0.21 2.69 1.28 −2.07  −2.51 Diff (1.78) (1.74) (1.74) (1.74) (1.42) (SE)/week 4 LS Mean −0.47 −2.44  0.11 0.27 −3.51  −5.2  (SE)/week 8 (1.34) (1.9)  (1.87) (1.87)(1.87) (1.32) LS Mean −1.97  0.58 0.74 −3.04  −4.73  Diff (2.35) (2.31)(2.3) (2.3)   (1.88)* (SE)/week 8 change LS Mean −0.09  −0.06  0.21 0.01−0.33  −0.41  (SE)/week 4 (0.12) (0.17) (0.16) (0.16) (0.16) (0.11) LSMean 0.02 0.3  0.09 −0.25  −0.33  Diff (0.2)  (0.2)  (0.2)  (0.2) (0.16) (SE)/week 4 LS Mean −0.05  −0.26  −0.03  −0.07  −0.41  −0.62 (SE)/week 8 (0.15) (0.22) (0.21) (0.21) (0.21) (0.15) LS Mean −0.21 0.02 −0.02  −0.37  −0.58  Diff (0.27) (0.26) (0.26) (0.26)  (0.22)*(SE)/week 8 Total Bone LS Mean 0.05 −0.22  −0.48  −0.04  0.47 0.55Mineral (SE)/week 4 (0.25) (0.36) (0.35) (0.36) (0.36) (0.25) Density(BMD) Mass (g/cm2)/% change LS Mean −0.27  −0.53  −0.09  0.42 0.5  Diff(0.43) (0.43) (0.44) (0.44) (0.35) (SE)/week 4 LS Mean −0.01  −0.47 −0.72  −0.08  −0.22  0.16 (SE)/week 8 (0.28) (0.4)  (0.4)  (0.4)  (0.41)(0.28) LS Mean −0.46 −0.71  −0.07  −0.21  0.17 Diff (0.49) (0.49) (0.5) (0.5)  (0.4)  (SE)/week 8 change LS Mean 0   −0.002 −0.005 0    0.005 0.006 (SE)/week 4  (0.003)  (0.004)  (0.004)  (0.004)  (0.004)  (0.003)LS Mean 0   −0.01  0   0   0.01 Diff (0)   (0)   (0)   (0)   (0)  (SE)/week 4 LS Mean 0   −0.006 −0.008 0   −0.002  0.002 (SE)/week 8 (0.003)  (0.004)  (0.004)  (0.004)  (0.004)  (0.003) LS Mean −0.01 −0.01  0   0   0   Diff (0.01) (0.01) (0.01) (0.01) (0)   (SE)/week 8Total Bone LS Mean 0.1  −0.16  −0.23  0.25 −0.19  0.62 Mineral (SE)/week4 (0.28) (0.4)  (0.4)  (0.4)  (0.4)  (0.28) Content (BMC) Mass (kg)/ %change LS Mean −0.26  −0.33  0.15 −0.29  0.52 Diff (0.49) (0.49) (0.49)(0.49) (0.4)  (SE)/week 4 LS Mean −0.42  −0.27  −0.48  −0.08  −0.56 0.65 (SE)/week 8 (0.26) (0.36) (0.36) (0.36) (0.36) (0.25) LS Mean 0.16−0.05  0.34 −0.14  1.07 Diff (0.45) (0.44) (0.44) (0.44)  (0.36)*(SE)/week 8 change LS Mean  0.002 −0.003 −0.005  0.006 −0.003  0.014(SE)/week 4  (0.006)  (0.009)  (0.009)  (0.009)  (0.009)  (0.006) LSMean −0.01  −0.01  0   −0.01  0.01 Diff (0.01) (0.01) (0.01) (0.01)(0.01) (SE)/week 4 LS Mean −0.009 −0.007 −0.011 −0.001 −0.012  0.014(SE)/week 8  (0.006)  (0.008)  (0.008)  (0.008)  (0.008)  (0.006) LSMean 0   0   0.01 0   0.02 Diff (0.01) (0.01) (0.01) (0.01)  (0.01)*(SE)/week 8 T-score for LS Mean −18.26  −51.19  −2.95  −6.66  −39.76 −0.9  Total (SE)/week 4 (18.01)  (25.74)  (25.45)  (25.7)  (25.87) (17.96)  Body/% change LS Mean −32.93  15.31  11.6  −21.5  17.37  Diff(31.23)  (31.1)  (31.56)  (31.73)  (25.43)  (SE)/week 4 LS Mean −20.87 −58.54  3.83 5.16 23.12  8.49 (SE)/week 8 (17.94)  (25.64)  (25.35) (25.6)  (25.77)  (17.89)  LS Mean −37.67 24.71  26.04  44   29.36  Diff(31.11)  (30.98)  (31.44)  (31.61)  (25.33)  (SE)/week 8 change LS Mean0.01 −0.03  −0.07  0   0.06 0.08 (SE)/week 4 (0.03) (0.05) (0.05) (0.05)(0.05) (0.03) LS Mean −0.04  −0.07  −0.01  0.06 0.07 Diff (0.06) (0.06)(0.06) (0.06) (0.05) (SE)/week 4 LS Mean 0   −0.07  −0.1  −0.01  −0.03 0.03 (SE)/week 8 (0.04) (0.05) (0.05) (0.05) (0.06) (0.04) LS Mean−0.07  −0.1  −0.01  −0.03  0.03 Diff (0.07) (0.07) (0.07) (0.07) (0.05)(SE)/week 8 Note: Least-squares (LS) means, standard errors (SE) andp-value taken from ANCOVA. The model includes baseline measurement ascovariate and the treatment as fixed factor. *indicated p-value < 0.05;**indicated p-value < 0.001.

Compared with placebo, R2477+R1033 combination significantly increasedthigh muscle volume and total lean mass in medium and high dose groups(FIGS. 8 and 9), significantly increased appendicular lean body mass inall dose groups (FIG. 10) and significantly decreased total fat mass aswell as android fat mass in the high dose group (Table 10). R2477+R1033,in the high dose group, significantly increased all of the majormeasures of muscle volume and lean mass compared with placebo, at bothWeek 4 and Week 8; the effects at Week 4 were generally less pronouncedthan at Week 8 (Table 10). At Week 8, the high dose anti-Activin AR2477+anti-GDF8 R1033 group exhibited increased % change in total bonemineral content from placebo, as measured by DXA, while total bonemineral density did not change (Table 10).

Safety

All treatment emergent adverse events (TEAEs) were mild to moderate inseverity except one severe TEAE of ‘radius fracture’ reported by aplacebo subject. There were no serious adverse events, no deaths, and nodiscontinuations due to TEAEs. Headache was the most frequent TEAE ineach of the treatment groups, occurring in 58.3% of all study subjectsand in 50% of placebo subjects. Muscle spasms, nausea and mouthulceration were the other frequent TEAEs in REGN2477+REGN1033 groupsthat occurred in 25% or more of subjects in the combination R2477+R1033groups; these TEAEs occurred less frequently in the placebo group, butthere does not appear to be any clear dose-response relationship. Therewere no clear signals of bleeding or diarrhea, adverse events that havebeen associated with blockade of activin receptors. One TEAE of anosebleed (preferred term of epistaxis) occurred in a R2477+R1033 dosegroup—it resolved after 9 minutes.

Review of Potentially Clinically Significant Values (PCSVs) revealed nosignificant differences between REGN2477+REGN1033 and Placebo in Labs,Vital signs, and ECG that would indicate negative effects ofREGN2477+REGN1033. Within laboratory, vital sign and ECG categories,there were 0-2 subjects with PCSVs in the combined REGN2477+REGN1033dose groups (N of 24); however the percentage of subjects with PCSVs wasequal to or lower than that found in the placebo group. There were notreatment-emergent PCSVs related to liver function tests.

Conclusions

In healthy postmenopausal women, single intravenous doses ofREGN2477+REGN1033 increased thigh muscle volume, total lean mass, andappendicular lean body mass. One surprising finding was the uniformityof the thigh muscle changes: all of the individuals exposed to thecombination exhibited an increase in thigh muscle volume, as shown inFIG. 4. In addition, single intravenous doses of REGN2477+REGN1033decreased total fat, and in particular, android fat mass. Treatment withREGN1033 alone increased thigh muscle volume.

In general, REGN2477, REGN1033, and REGN2477+REGN1033 in this clinicalstudy were considered to have an acceptable safety profile and were welltolerated. There were no serious adverse events.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1. A method for altering the body composition of a subject comprisingadministering a first composition comprising an effective amount of aGDF8 inhibitor and a second composition comprising an effective amountof an Activin A inhibitor to the subject.
 2. The method of claim 1,wherein the altering of the body composition comprises inducing areduction in fat mass in the subject comprising administering the firstcomposition comprising an effective amount of a GDF8 inhibitor and thesecond composition comprising an effective amount of an Activin Ainhibitor to the subject.
 3. The method of claim 2, wherein the alteringof the body composition comprises inducing an increase in muscle mass inthe subject, the method comprising administering the first compositioncomprising an effective amount of the GDF8 inhibitor and the secondcomposition comprising an effective amount of the Activin A inhibitor.4. A method for altering the body composition of a subject comprisingadministering a composition comprising an effective amount of a GDF8inhibitor and an effective amount of an Activin A inhibitor to thesubject.
 5. The method of claim 4, wherein the altering of the bodycomposition comprises inducing a reduction in fat mass in a subjectcomprising administering the composition comprising an effective amountof the GDF8 inhibitor and an effective amount of the Activin A inhibitorto the subject.
 6. The method of claim 5, wherein the altering of thebody composition comprises inducing an increase in muscle mass in thesubject, the method comprising administering to the subject in needthereof the composition comprising an effective amount of the GDF8inhibitor and an effective amount of the Activin A inhibitor.
 7. Themethod of claim 1, wherein the effective amount of a GDF8 inhibitorcomprises a dosing regimen selected from the group consisting of atleast 0.01 mg/kg to about 10 gm/kg, 1 mg/kg to about 1 gm/kg, and 10mg/kg to 100 mg/kg.
 8. The method of claim 7, wherein the effectiveamount of an Activin A inhibitor comprises a dosing regimen selectedfrom the group consisting of at least 0.01 mg/kg to about 10 gm/kg, 1mg/kg to about 1 gm/kg, and 10 mg/kg to 100 mg/kg.
 9. The method ofclaim 1, wherein the effective amount of a GDF8 inhibitor comprises adosing regimen selected from a group consisting of a single dose ofabout 0.01 to about 20 mg/kg body weight, about 0.1 to about 10 mg/kgbody weight, and about 0.1 to about 5 mg/kg body weight.
 10. The methodof claim 9, wherein the effective amount of an Activin A inhibitorcomprises a dosing regimen selected from a group consisting of a singledose of about 0.01 to about 20 mg/kg body weight, about 0.1 to about 10mg/kg body weight, and about 0.1 to about 5 mg/kg body weight.
 11. Themethod of claim 10, wherein the effective amount of the GDF8 inhibitoris 6 mg/kg body weight of the subject.
 12. The method of claim 11,wherein the effective amount of the Activin A inhibitor is 3 mg/kg or 10mg/kg body weight of the subject.
 13. The method of claim 1, wherein thefirst composition is formulated for intravenous, subcutaneous, or oraladministration.
 14. The method of claim 1, wherein the secondcomposition is formulated for intravenous, subcutaneous, or oraladministration.
 15. The method of claim 1, wherein the first and secondcompositions are combined into a third composition prior toadministration.
 16. The method of claim 15, wherein the thirdcomposition is formulated for intravenous, subcutaneous, or oraladministration.
 17. The method of claim 5, wherein the composition isformulated for intravenous, subcutaneous, or oral administration. 18.The method of claim 2, further comprising measuring total fat massand/or android fat mass in the subject before administration.
 19. Themethod of claim 18, further comprising measuring total fat mass and/orandroid fat mass in the subject after administration, and administeringthe composition until the subject has a reduction in total fat massand/or android fat mass of at least about 2% to 8%, at least about 2.5%to 6%, at least about 3% to 4%, or at least about 3.5%.
 20. The methodof claim 1, wherein the GDF8 inhibitor is an isolated antibody or anantigen-binding fragment thereof that specifically binds to GDF8. 21.The method of claim 20, wherein the antibody or antigen-binding fragmentthat specifically binds GDF8 comprises the heavy chain complementaritydetermining regions (HCDRs) of a heavy chain variable region (HCVR)comprising SEQ ID NO:360, and the light chain complementaritydetermining regions (LCDRs) of a light chain variable region (LCVR)comprising SEQ ID NO:368.
 22. The method of claim 20, wherein theantibody or antigen-binding fragment that specifically binds GDF8comprises three HCDRs comprising SEQ ID NO:362, SEQ ID NO:364, and SEQID NO:366, and three LCDRs comprising SEQ ID NO:370, SEQ ID NO:372, andSEQ ID NO:374.
 23. The method of claim 1, wherein the Activin Ainhibitor is an isolated antibody or antigen-binding fragment thereofthat specifically binds Activin A.
 24. The method of claim 23, whereinthe antibody or antigen-binding fragment that specifically binds ActivinA comprises the heavy chain complementarity determining regions (HCDRs)of a heavy chain variable region (HCVR) comprising SEQ ID NO:553, andthe light chain complementarity determining regions (LCDRs) of a lightchain variable region (LCVR) comprising SEQ ID NO:537.
 25. The method ofclaim 23, wherein the antibody or antigen-binding fragment thatspecifically binds Activin A comprises three HCDRs comprising SEQ IDNO:555, SEQ ID NO:557, and SEQ ID NO:559, and three LCDRs comprising SEQID NO:539, SEQ ID NO:541, and SEQ ID NO:543.
 26. The method of any oneof claim 1, wherein the Activin A inhibitor is in an amount selectedfrom the group consisting of between 100% to 200% of the amount of theGDF8 inhibitor, between 100% and 250% of the amount of the GDF8inhibitor, between 100% and 300% of the amount of the GDF8 inhibitor,and between 100% and 400% by weight of the amount of the GDF8 inhibitor.27. The method of claim 26, wherein the amount of the Activin Ainhibitor is about 1.5 to 2.0 times as large by weight as the amount ofthe GDF8 inhibitor.
 28. The method of claim 2, wherein a reduction offat mass in the subject is a reduction in total fat mass as measured byDXA (Dual-energy X-ray absorptiometry).
 29. The method of claim 2,wherein a reduction of fat mass in the subject is a reduction in androidfat mass as measured by DXA (Dual-energy X-ray absorptiometry).
 30. Themethod of claim 1, wherein the subject experiences an increase in musclevolume after administration.
 31. The method of claim 1, wherein thesubject does not have a muscle wasting condition or disease.
 32. Amethod for treating a disease or disorder characterized by increased fatmass, wherein the method comprises administrating to a subject the GDF8inhibitor and an Activin A inhibitor.
 33. (canceled)
 34. Anon-therapeutic method for decreasing fat mass in a subject, the methodcomprising administering to the subject an Activin A inhibitor and aGDF8 inhibitor.